Pyrimidinyl tyrosine kinase inhibitors

ABSTRACT

The present invention provides compounds and compositions thereof which are useful as inhibitors of Bruton&#39;s tyrosine kinase and which exhibit desirable characteristics for the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/911,546, filed on Mar. 5, 2018 (now allowed), which is a continuationof U.S. application Ser. No. 15/196,389, filed on Jun. 29, 2016 (nowU.S. Pat. No. 9,944,622), which is a continuation of U.S. applicationSer. No. 14/406,315, filed Dec. 8, 2014 (now U.S. Pat. No. 9,394,277),which is a U.S. National Phase application, filed under 35 U.S.C. § 371,of International Application No. PCT/US2013/044800, filed Jun. 7, 2013,which claims priority to U.S. provisional application Ser. No.61/657,360, filed Jun. 8, 2012, the entire contents of each of which arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The Tec kinases are non-receptor tyrosine kinases including: Tec(tyrosine kinase expressed in hepatocellular carcinoma), Btk (Bruton'styrosine kinase), Itk (interleukin-2 (IL-2)-inducible T-cell kinase;also known as Emt or Tsk), Rlk (resting lymphocyte kinase; also known asTxk), Lck (lymphocyte-specific protein tyrosine kinase), and Bmx(bone-marrow tyrosine kinase gene on chromosome X; also known as Etk)).These kinases are primarily expressed in haematopoietic cells, althoughexpression of Bmx and Tec has been detected in endothelial and livercells. Tec kinases (Itk, Rlk and Tec) are expressed in T cells and areall activated downstream of the T-cell receptor (TCR). Btk is adownstream mediator of B cell receptor (BCR) signaling which is involvedin regulating B cell activation, proliferation, and differentiation.More specifically, Btk contains a PH domain that bindsphosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 binding inducesBtk to phosphorylate phospholipase C (PLC), which in turn hydrolyzesPIP2 to produce two secondary messengers, inositol triphosphate (IP3)and diacylglycerol (DAG), which activate protein kinase PKC, which theninduces additional B-cell signaling. Mutations that disable Btkenzymatic activity result in XLA syndrome (X-linked agammaglobulinemia),a primary immunodeficiency. Given the critical roles which Tec kinasesplay in both B-cell and T-cell signaling, Tec kinases are targets ofinterest for autoimmune disorders.

Consequently, there is a great need in the art for effective inhibitorsfor Tec kinases such as Btk. The present invention fulfills these andother needs.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides a compound offormula I

or a pharmaceutically acceptable salt thereof, wherein R¹ and Ring A areas defined and described herein. Such compounds are inhibitors of theTec kinase family, including Btk. Accordingly, provided compounds can beused in a variety of methods including in vitro screening and activityassays as well as in vivo pre-clinical, clinical, and therapeuticsettings, as described in detail herein.

In certain embodiments, the present invention provides pharmaceuticalformulations comprising provided compounds.

In certain embodiments, the present invention provides a method ofdecreasing enzymatic activity of Btk. In some embodiments, such methodsinclude contacting Btk with an effective amount of a Btk inhibitor.

In certain embodiments, the present invention provides a method oftreating a disorder responsive to Btk inhibition in a subject in needthereof. Such disorders and methods are described in detail herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof;wherein:

-   -   each R¹ is independently hydrogen, an optionally substituted        C₁₋₆ aliphatic group, an optionally substituted 3-7 membered        monocyclic heterocyclic group, or an optionally substituted        heterocyclylalkyl group having 3-7 carbon atoms and 1-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;        -   or two R¹ groups are taken together with their intervening            atoms to form an optionally substituted 3-7 membered            saturated or partially unsaturated monocyclic heterocyclic            ring having 1-2 heteroatoms independently selected from            nitrogen, oxygen, or sulfur;    -   wherein optionally substituted groups may be substituted with        halogen, —NO₂, —CN, —OR, —SR, —N(R)₂, —C(O)R, —CO₂R,        —N(R)C(O)OR, —C(O)N(R)₂, —OC(O)R, —N(R)C(O)R, —S(O)R, —S(O)₂R,        or —S(O)₂N(R)₂;    -   each R is independently hydrogen or C₁₋₆ aliphatic;        -   or two R groups attached to the same nitrogen are taken            together with their intervening atoms to form an optionally            substituted 3-7 membered saturated or partially unsaturated            monocyclic heterocyclic ring having 1-2 heteroatoms, in            which any second heteroatom is independently selected from            nitrogen, oxygen, or sulfur;    -   Ring A is

-   -   R² is —Cl or —F; and    -   R³ is —CF₃, —OCF₃, or —F.

Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. Suitable aliphaticgroups include, but are not limited to, linear or branched, substitutedor unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof suchas (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “cycloaliphatic” (or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule.

As used herein, the terms “heterocycle,” “heterocyclyl,” and“heterocyclic ring” are used interchangeably and refer to a stable 3- to7-membered monocyclic heterocyclic moiety that is either saturated orpartially unsaturated, and having, in addition to carbon atoms, one ormore, preferably one to four, heteroatoms, as defined above. When usedin reference to a ring atom of a heterocycle, the term “nitrogen”includes a substituted nitrogen. As an example, in a saturated orpartially unsaturated ring having 0-3 heteroatoms selected from oxygen,sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as inN-substituted pyrrolidinyl). The term “heterocyclylalkyl” refers to analkyl group substituted by a heterocyclyl, wherein the alkyl andheterocyclyl portions independently are optionally substituted.

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference.

In certain embodiments, the neutral forms of the compounds areregenerated by contacting the salt with a base or acid and isolating theparent compound in the conventional manner. In some embodiments, theparent form of the compound differs from the various salt forms incertain physical properties, such as solubility in polar solvents.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

Compounds

As described above, in certain embodiments the present inventionprovides a compound of formula I:

of a pharmaceutically acceptable salt thereof, wherein R¹ and Ring A areas defined and described herein.

Compounds of formula I have unexpectedly been found to exhibitadvantageous properties over known inhibitors of Btk. In certainembodiments, compounds of formula I have increased potency. Withoutwishing to be bound by any particular theory, it is believed thatcompounds disclosed herein possess improved potency as Btk inhibitors,an improved off-target profile as measured by hERG inhibition or PXRinduction assays, or a combination thereof. Experimental data showingsuch advantageous properties is provided in the ensuing Examples.

In some embodiments, both R¹ are hydrogen. In some embodiments, each R¹is independently C₁₋₆ aliphatic. In some embodiments, each R¹ isindependently C₁₋₅ aliphatic. In some embodiments, each R¹ isindependently C₁₋₄ aliphatic. In some embodiments, each R¹ isindependently C₁₋₃ aliphatic. In some embodiments, each R¹ isindependently C₁₋₂ aliphatic. In some embodiments, both R¹ are methyl.

In some embodiments, each R¹ is independently hydrogen or C₁₋₆aliphatic. In some embodiments, each R¹ is independently hydrogen orC₁₋₅ aliphatic. In some embodiments, each R¹ is independently hydrogenor C₁₋₄ aliphatic. In some embodiments, each R¹ is independentlyhydrogen or C₁₋₃ aliphatic. In some embodiments, each R¹ isindependently hydrogen or C₁₋₂ aliphatic. In some embodiments, each R¹is independently hydrogen or methyl.

In some embodiments, one R¹ is hydrogen or and the other R¹ is C₁₋₆aliphatic. In some embodiments, one R¹ is hydrogen and the other R¹ ismethyl. In some embodiments, one R¹ is hydrogen and the other R¹ isethyl. In some embodiments, one R¹ is hydrogen and the other R¹ is C₁₋₆(cycloalkyl)alkyl. In some embodiments, one R¹ is hydrogen and the otherR¹ is C₁₋₆ (cycloalkyl).

In some embodiments, one R¹ is hydrogen and the other R¹ is C₁₋₆aliphatic optionally substituted with —OR, wherein R is hydrogen or C₁₋₆aliphatic.

In some embodiments, one R¹ is hydrogen and the other R¹ is aheterocyclylalkyl group having 3-7 carbon atoms and 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, one R¹ is hydrogen and the other R¹ is an optionallysubstituted 3-7 membered monocyclic heterocycle.

In some embodiments, two R¹ groups are taken together with theirintervening atoms to form an optionally substituted 3-5 memberedsaturated or partially unsaturated monocyclic heterocyclic ring having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, two R¹ groups are taken together with theirintervening atoms to form an optionally substituted piperazine ring.

As described above, Ring A is

In certain embodiments, R² is —Cl. In other embodiments, R² is —F. Insome embodiments, R³ is —CF₃. In some embodiments, R³ is —OCF₃. In someembodiments, R³ is —F.

In certain embodiments, Ring A is selected from the group consisting of:

In some embodiments, in compounds described herein there is a transstereochemical relationship between the piperidine substituent bearingthe carboxamide group and the piperidine substituent bearing the lactamgroup.

In some embodiments, the present invention provides a compound offormula II-a:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,and R³ is as defined above and described in classes and subclassesherein.

In some embodiments, the present invention provides a compound offormula II-b:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,and R³ is as defined above and described in classes and subclassesherein.

In some embodiments, the present invention provides a compound offormula III:

or a pharmaceutically acceptable salt thereof, wherein each of R² and R³is as defined above and described in classes and subclasses herein.

In some embodiments, the present invention provides a compound offormula IV:

or a pharmaceutically acceptable salt thereof, wherein each of R¹ and R³is as defined above and described in classes and subclasses herein. Insome embodiments, both R¹ are hydrogen. In some embodiments, one R¹ ishydrogen and the other R¹ is methyl.

In some embodiments, a provided compound is a compound depicted in Table1, below, or a pharmaceutically acceptable salt thereof.

Table 1—Selected compounds of formula I.

Compounds of the invention are synthesized by an appropriate combinationof generally well known synthetic methods. Techniques useful insynthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention. However, thediscussion is not intended to define the scope of reactions or reactionsequences that are useful in preparing the compounds of the presentinvention.

Compounds of formula I may be generally prepared according to Scheme 1.

Compounds of formula I may also be generally prepared according toSchemes 2, 3, 3a, 4, 4a, 5, or 5a.

The PG, PG₁, PG₂, and PG₃ groups of compounds in Schemes 1 through 5aare each independently a suitable protecting group. Such ester and amineprotecting groups are known in the art and are described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. In some embodiments, a protectinggroup is a Boc group.

In certain embodiments, each of the synthetic steps in Schemes 1 through5a may be performed sequentially with isolation of each intermediateperformed after each step. Alternatively, each of the steps as depictedin Schemes 1, 2, 3, and 4 above, may be performed in a manner whereby noisolation of one or more intermediates is performed.

In certain embodiments, all the steps of the aforementioned synthesismay be performed to prepare the desired final product. In otherembodiments, two, three, four, five, or more sequential steps may beperformed to prepare an intermediate or the desired final product.

Uses, Formulation and Administration

In certain embodiments, compounds of the present invention are for usein medicine. In some embodiments, the present invention provides methodof decreasing enzymatic activity of a kinase in the Tec kinase family(e.g., Tec, Btk, Itk, Txk, Lck, and Bmx). In some embodiments, suchmethods include contacting a kinase of the Tec kinase family with aneffective amount of a Tec kinase family inhibitor. Therefore, thepresent invention further provides methods of inhibiting Tec kinasefamily enzymatic activity by contacting a Tec kinase family member witha Tec kinase family inhibitor of the present invention. As used herein,the term “Tec kinase family member” refers to any non-receptor tyrosinekinase in the Tec kinase family. In some embodiments, Tec kinase familymembers are Tec, Btk, Itk, Txk, Lck, and Bmx.

In some embodiments, the present invention provides methods ofdecreasing Btk enzymatic activity. In some embodiments, such methodsinclude contacting a Btk with an effective amount of a Btk inhibitor.Therefore, the present invention further provides methods of inhibitingBtk enzymatic activity by contacting a Btk with a Btk inhibitor of thepresent invention.

Btk enzymatic activity, as used herein, refers to Btk kinase enzymaticactivity. For example, where Btk enzymatic activity is decreased, PIP3binding and/or phosphorylation of PLCγ is decreased. In someembodiments, the half maximal inhibitory concentration (IC₅₀) of the Btkinhibitor against Btk is less than 1 M. In some embodiments, the IC₅₀ ofthe Btk inhibitor against Btk is less than 500 nM. In some embodiments,the IC₅₀ of the Btk inhibitor against Btk is less than 100 nM. In someembodiments, the IC₅₀ of the Btk inhibitor against Btk is less than 10nM. In some embodiments, the IC₅₀ of the Btk inhibitor against Btk isless than 1 nM. In some embodiments, the IC₅₀ of the Btk inhibitoragainst Btk is from 0.1 nM to 10 M. In some embodiments, the IC₅₀ of theBtk inhibitor against Btk is from 0.1 nM to 1 M. In some embodiments,the IC₅₀ of the Btk inhibitor against Btk is from 0.1 nM to 100 nM. Insome embodiments, the IC₅₀ of the Btk inhibitor against Btk is from 0.1nM to 10 nM.

In some embodiments, inhibitors of such Tec kinases are useful for thetreatment of diseases and disorders that may be alleviated by inhibiting(i.e., decreasing) enzymatic activity of one or more Tec kinases. Thecompounds of the invention are effective inhibitors of Tec familykinases and would thus be useful in treating diseases associated withthe activity of one or more of the Tec family kinases. The term“diseases” means diseases, syndromes, or disease symptoms. Thus, thepresent invention provides methods of treating autoimmune disorders,inflammatory disorders, and cancers in a subject in need thereof. Suchmethods include administering to the subject a therapeutically effectiveamount of an inhibitor of Tec, Btk, Itk, Txk, Lck, and/or Bmx kinase.

The term “autoimmune disorders” includes diseases or disorders involvinginappropriate immune response against native antigens, such as acutedisseminated encephalomyelitis (ADEM), Addison's disease, alopeciaareata, antiphospholipid antibody syndrome (APS), hemolytic anemia,autoimmune hepatitis, bullous pemphigoid (BP), Coeliac disease,dermatomyositis, diabetes mellitus type 1, Good Pasture's syndrome,Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease,idiopathic thrombocytopenic purpura, lupus or systemic lupuserythematosus (SLE), mixed connective tissue disease, multiplesclerosis, myasthenia gravis, pemphigus vulgaris, hemophilia withinhibitors, pernicious anaemia, polymyositis, primary biliary cirrhosis,Sjögren's syndrome, temporal arteritis, and Wegener's granulomatosis.The term “inflammatory disorders” includes diseases or disordersinvolving acute or chronic inflammation such as allergies, asthma (e.g.,allergic asthma), atopic dermatitis, prostatitis, glomerulonephritis,pelvic inflammatory disease (PID), inflammatory bowel disease (IBD,e.g., Crohn's disease, ulcerative colitis), reperfusion injury,rheumatoid arthritis, transplant rejection (including transplantpatients with a positive cross-match) and vasculitis. In certainembodiments, the present invention provides methods of treating disease,disorders, or conditions that approved for treatment with rituximab (amonoclonal antibody against CD20), including non-Hodgkin's lymphoma(NHL), chronic lymphocytic leukemia (CLL), RA, Wegener's granulomatosis(WG), and microscopic polyangiitis (MPA). In some embodiments, thepresent invention provides a method of treating rheumatoid arthritis(RA), SLE, or atopic dermatitis using compounds disclosed herein.

The term “cancer” includes diseases or disorders involving abnormal cellgrowth and/or proliferation, such as glioma, thyroid carcinoma, breastcarcinoma, lung cancer (e.g. small-cell lung carcinoma, non-small-celllung carcinoma), gastric carcinoma, gastrointestinal stromal tumors,pancreatic carcinoma, bile duct carcinoma, ovarian carcinoma,endometrial carcinoma, prostate carcinoma, renal cell carcinoma,lymphoma (e.g., anaplastic large-cell lymphoma), leukemia (e.g. acutemyeloid leukemia, T-cell leukemia, chronic lymphocytic leukemia),multiple myeloma, malignant mesothelioma, malignant melanoma, and coloncancer (e.g. microsatellite instability-high colorectal cancer). In someembodiments, the present invention provides a method of treatingleukemia or lymphoma.

The term “subject,” as used herein, refers to a mammal to whom apharmaceutical composition is administered. Exemplary subjects includehumans, as well as veterinary and laboratory animals such as horses,pigs, cattle, dogs, cats, rabbits, rats, mice, and aquatic mammals.

Selected Indications and B Cell Inhibition

As described above, provided compounds are useful for the treatment ofdisease, including RA and SLE. As described in more detail below, thesediseases are affiliated with B cells. Thus, the present disclosureencompasses the recognition that provided compounds are useful astherapeutics for these and other indications.

Dysregulation of the immune system is central to the pathogenesis(Panayi G S, et al. Rheum Dis Clin North Am 2001; 27:317-334) of RA.While most of the infiltrating leukocytes in the synovium are Tlymphocytes (primarily activated CD4+ T cells) and cells ofmonocyte/macrophage origin (which release pro-inflammatory cytokinessuch as IL-1, TNF-alpha and IL-6 and proteolytic enzymes includingcollagenases and metalloproteinases), B-cells and plasma cells are alsofound in the synovial fluid (Zhang Z, Bridges S L. Rheum Dis Clin NorthAm 2001; 27:335-353). A clear role for B cells and their associatedeffector functions in RA have been demonstrated by the efficacy ofrituximab, a selective B cell depleting therapeutic, which is approvedfor treatment of RA (Cohen S B, et al.; REFLEX Trial Group. ArthritisRheum. 2006 September; 54(9):2793-806).

Although the etiology of SLE is not fully understood, pathogenicautoantibodies and deposition of immune complexes are felt to becritical to the development of widespread tissue damage (Klippel J H, etal. Primer on the rheumatic diseases. Atlanta: Arthritis Foundation;2001). Autoantibody and immune-complex mediated activation can bestudied by measuring inhibition of macrophage activation by macrophagesstimulated through Fc receptors (see exemplification—FcγR activation ofprimary human macrophages). Loss of tolerance to self-antigensultimately lead to the stimulation of B cells to produce auto-antibodiesoften directed against nuclear or cytoplasmic components. Antibodiesagainst nuclear components (anti-nuclear antibodies [ANA]) targetnuclear antigens including DNA (typically double-stranded DNA [dsDNA]),RNA, histones and small nuclear ribonucleoproteins. These antibodiescombine with self-antigens forming immune complexes which deposit intissues, incite inflammatory reactions and lead to tissue injury. Inaddition to their roles in pathogenic autoantibody production, B cellsalso function as antigen-presenting cells (APCs) to T-cells thus playinga role in the initiation of an antigen-specific response. Given thecentral role of the humoral arm of the immune system in the pathogenesisof SLE, B cells or the B-cell pathway represent desirable therapeutictargets. Belimumab, a monoclonal antibody recently approved for SLE,blocks the binding BAFF to its receptors that are expressed B cells.These receptors serve to activate and potentiate the survival of B cellsconsistent with a reduction of circulating B cells observed followingtreatment with belimumab. See also Chan O T, et al. Immunol Rev. 1999b;169:107-121; Navarra S V, et al. Lancet. 2011 Feb. 26; 377(9767):721-31;Furie R, et al. Arthritis Rheum. 2011 December; 63(12):3918-30. The roleof B cells and myeloid lineage cells in autoimmune diseases such as SLEis further supported by a recent publication which describes efficacy ina preclinical SLE animal model when mice are treated with a smallmolecule irreversible Btk inhibitor (Honigberg, L. A. PNAS. 2010; 107:13075).

Combinations

In certain embodiments, a compound of the present invention isadministered in combination with another agent. In some embodiments, acompound of the present invention is useful for treating RA and isadministered in combination with a disease-modifying antirheumatic drugs(DMARD), including without limitation: methotrexate, abatacept,azathioprine, certolizumab, chloroquine and hydroxychloroquine,cyclosporin, D-penicillamine, adalimumab, etanercept, golimumab, goldsalts (including auranofin and sodium aurothiomalate), infliximab,leflunomide, minocycline, rituximab, sulfasalazine, tocilizumab, orcombinations thereof. In some embodiments, a compound of the presentinvention is administered in combination with a NSAID or corticosteroid.In some embodiments, a compound of the present invention is useful fortreating SLE and is administered in combination with an agent for thetreatment of SLE, including without limitation: corticosteroids,antimalarials, belimumab, mycophenolate mofetil (MMF) or mycophenolatesodium, azathioprine, or combinations thereof. In some embodiments, acompound of the present invention is useful for treating atopicdermatitis and is administered in combination with a topical agent forthe treatment of atopic dermatitis, including without limitation:topical steroids, tacrolimus, methotrexate, mometasone furoate (MMF),azathioprine, retinoids, or combinations thereof.

Assays

To develop useful Tec kinase family inhibitors, candidate inhibitorscapable of decreasing Tec kinase family enzymatic activity may beidentified in vitro. The activity of the inhibitor compounds can beassayed utilizing methods known in the art and/or those methodspresented herein.

Compounds that decrease Tec kinase family members' enzymatic activitymay be identified and tested using a biologically active Tec kinasefamily member, either recombinant or naturally occurring. Tec kinasescan be found in native cells, isolated in vitro, or co-expressed orexpressed in a cell. Measuring the reduction in the Tec kinase familymember enzymatic activity in the presence of an inhibitor relative tothe activity in the absence of the inhibitor may be performed using avariety of methods known in the art, such as the POLYGAT-LS assaysdescribed below in the Examples. Other methods for assaying the activityof Btk and other Tec kinases are known in the art. The selection ofappropriate assay methods is well within the capabilities of those ofskill in the art.

Once compounds are identified that are capable of reducing Tec kinasefamily members' enzymatic activity, the compounds may be further testedfor their ability to selectively inhibit a Tec kinase family memberrelative to other enzymes.

Compounds may be further tested in cell models or animal models fortheir ability to cause a detectable changes in phenotype related to aTec kinase family member activity. In addition to cell cultures, animalmodels may be used to test Tec kinase family member inhibitors for theirability to treat autoimmune disorders, inflammatory disorders, or cancerin an animal model.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions comprising a compound of formula I or a compound of formulaI in combination with a pharmaceutically acceptable excipient (e.g.,carrier).

The pharmaceutical compositions include optical isomers, diastereomers,or pharmaceutically acceptable salts of the inhibitors disclosed herein.The compound of formula I included in the pharmaceutical composition maybe covalently attached to a carrier moiety, as described above.Alternatively, the compound of formula I included in the pharmaceuticalcomposition is not covalently linked to a carrier moiety.

A “pharmaceutically acceptable carrier,” as used herein refers topharmaceutical excipients, for example, pharmaceutically,physiologically, acceptable organic or inorganic carrier substancessuitable for enteral or parenteral application that do not deleteriouslyreact with the active agent. Suitable pharmaceutically acceptablecarriers include water, salt solutions (such as Ringer's solution),alcohols, oils, gelatins, and carbohydrates such as lactose, amylose orstarch, fatty acid esters, hydroxymethycellulose, and polyvinylpyrrolidine. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention.

The compounds of the invention can be administered alone or can beco-administered to the subject. Co-administration is meant to includesimultaneous or sequential administration of the compounds individuallyor in combination (more than one compound). The preparations can also becombined, when desired, with other active substances (e.g. to reducemetabolic degradation).

Compounds of the present invention can be prepared and administered in awide variety of oral, parenteral, and topical dosage forms. Thus, thecompounds of the present invention can be administered by injection(e.g. intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally). Also, the compounds describedherein can be administered by inhalation, for example, intranasally.Additionally, the compounds of the present invention can be administeredtransdermally. It is also envisioned that multiple routes ofadministration (e.g., intramuscular, oral, transdermal) can be used toadminister the compounds of the invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substance that may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid in a mixture with thefinely divided active component. In tablets, the active component ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% to 70% of the activecompound. Suitable carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures for the compounds of the invention are injectable, sterilesolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants, including suppositories. In particular, carriersfor parenteral administration include aqueous solutions of dextrose,saline, pure water, ethanol, glycerol, propylene glycol, peanut oil,sesame oil, polyoxyethylene-block polymers, and the like. Ampoules areconvenient unit dosages. The compounds of the invention can also beincorporated into liposomes or administered via transdermal pumps orpatches. Pharmaceutical admixtures suitable for use in the presentinvention include those described, for example, in PharmaceuticalSciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309, theteachings of both of which are hereby incorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60, and 80; Pluronic F-68,F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Suchco-solvents are typically employed at a level between about 0.01% andabout 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation, and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, and combinations of the foregoing. Such agents aretypically employed at a level between about 0.01% and about 2% byweight.

The compositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides, and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes.

Effective Dosages

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For example, when administered in methods to treat cancer, suchcompositions will contain an amount of active ingredient effective toachieve the desired result (e.g. decreasing the number of cancer cellsin a subject).

The dosage and frequency (single or multiple doses) of compoundadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated (e.g., the disease responsive to Btk inhibition);presence of other diseases or other health-related problems; kind ofconcurrent treatment; and complications from any disease or treatmentregimen. Other therapeutic regimens or agents can be used in conjunctionwith the methods and compounds of the invention.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of decreasing kinase enzymatic activity as measured, forexample, using the methods described.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring kinase inhibition andadjusting the dosage upwards or downwards, as described above. Incertain embodiments, the administered dose is in the range of about 10mg to about 1000 mg per day, either once, twice, or more than twicedaily.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention, should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side effects. Generally, treatment is initiated with smallerdosages, which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. In some embodiments, thedosage range is 0.001% to 10% w/v. In some embodiments, the dosage rangeis 0.1% to 5% w/v.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Example 1 Synthetic of (3′R,4′S)-1′-tert-buty 4′-ethyl2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate

Preparation of ethyl 3-oxopiperidine-4-carboxylate intermediate. Ethyl1-benzyl-3-oxopiperidine-4-carboxylate 1 (15.0 g, 50.5 mmol, 1.0 equiv)was hydrogenated in the presence of 10% Pd/C (1.5 g) catalyst under H₂at atmospheric pressure in MeOH (250 mL) for 16 h. The catalyst wasfiltered off and the solvent was concentrated in vacuo to give ethyl3-oxopiperidine-4-carboxylate 2 as a light yellow solid (10.2 g, yield:98.0%). ESI-MS (M+H)⁺: 172.1. ¹H NMR (400 MHz, DMSO-d₆) δ: 4.23 (q, 2H),3.75 (s, 2H), 3.37 (s, 2H), 3.20-3.16 (m, 2H), 2.44 (t, 1H), 1.25 (t,3H).

Preparation of 1-tert-butyl 4-ethyl 3-oxopiperidine-1,4-dicarboxylate.Ethyl 3-oxopiperidine-4-carboxylate 2 (10.2 g, 60.0 mmol, 1.0 equiv) wasdissolved in dry MeOH (200 mL), and Et₃N (33.1 mL, 240 mmol, 4.0 equiv)was added. The mixture was stirred for 1 h and Boc₂O (19.5 g, 90.0 mmol,1.5 equiv) was added and stirred for 16 h. The solvent was concentratedin vacuo and the crude was purified by column chromatography (silica,petroleum ether/EtOAc=9:1) to give 1-tert-butyl 4-ethyl3-oxopiperidine-1,4-dicarboxylate 3 light yellow oil (11.5 g, yield:86%). ESI-MS (M+H−56)⁺: 216.0. ¹H NMR (400 MHz, CDCl₃) δ: 4.24 (q, 2H),4.03 (s, 2H), 3.49 (t, 2H), 2.33 (t, 2H), 1.47 (s, 9H), 1.31 (t, 3H).

Preparation of (S)-1-tert-butyl 4-ethyl3-((1-phenylethyl)amino)-5,6-dihydro pyridine-1,4-(2H)-dicarboxylate. Ina dry flask equipped with a Dean-stark trap and reflux condenser,1-tert-butyl 4-ethyl 3-oxopiperidine-1,4-dicarboxylate 3 (10.0 g, 37.0mmol, 1.1 equiv) was dissolved in toluene (100 mL).S-(−)-α-Methylbenzylamine (4.9 g, 40.5 mmol, 1.1 equiv) andp-toluenesulfonic acid monohydrate (0.7 g, 3.7 mmol, 0.1 equiv) wereadded and the mixture was heated to reflux for 16 h. The crude reactionmixture was concentrated in vacuo to give (S)-1-tert-butyl 4-ethyl3-((1-phenylethyl)amino)-5,6-dihydro pyridine-1,4(2H)-dicarboxylate 4(12.0 g, Y: 88%) as thick orange oil which was used in next step withoutfurther purification, ESI-MS (M+H)⁺: 375.2.

Preparation of 1-tert-butyl 4-ethyl3-(((S)1-phenylethyl)amino)-5,6-dihydro pyridine-1,4(2H)-dicarboxylate.1-tert-Butyl 4-ethyl3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 4 (11.2 g, 30.0mmol, 1.0 equiv) was dissolved in a mixture of CH₃CN (60 mL) and aceticacid (60 mL) and cooled to 0° C. NaBH(OAc)₃ (19.0 g, 90.0 mmol, 3.0equiv) was slowly added and the reaction mixture was allowed to stir for2 h at 0° C. Saturated NaHCO₃ was slowly added to neutralize thesolution to maintain the internal temperature of the flask below 10° C.The mixture was extracted with EtOAc (50 mL×3). The combined organiclayer was dried (Na₂SO₄), filtered, concentrated in vacuo, and thenpurified by column chromatography (silica, petroleum ether/EtOAc=9:1) togive 4-ethyl 3-(((S)1-phenylethyl)amino)-5,6-dihydropyridine-1,4(2H)-dicarboxylate 5 (8.2 g, Y: 73%) as light yellow oil.ESI-MS (M+H)⁺: 377.2. ¹H NMR (400 MHz, CD₃OD) δ: 7.31-7.22 (m, 5H), 4.20(q, 2H), 4.11-3.86 (m, 3H), 3.15 (s, 1H), 3.00-2.90 (m, 2H), 2.64 (d,2H), 1.87-1.85 (m, 1H), 1.68 (s, 1H), 1.50-1.25 (m, 15H).

Preparation of trans-1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate. The 1-tert-butyl 4-ethyl3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 5 (8.0 g, 21.2mmol, 1.0 equiv) was dissolved in dry EtOH (20 mL) under N₂. In aseparate flame-dried Schlenk flask was placed dry EtOH (150 mL), andsodium (0.450 g, 63.6 mmol, 3.0 equiv) was added portion-wise under N₂.The mixture was kept under N₂ and vented to remove evolved gases untilall of the sodium had dissolved. The clear solution of 1-tert-butyl4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate wasthen transferred to the NaOEt solution, and the mixture was stirred at80° C. under N₂ for 16 h. The solvent was removed under in vacuo, andbrine (150 mL) was added and the mixture was brought to pH=10 with 1 NNaOH and extracted with EtOAc (100 mL×3). The combined organic layerswere dried (Na₂SO₄) and concentrated in vacuo. The residue was purifiedby column chromatography (silica, petroleum ether/EtOAc=5:1) to give(trans)-1-tert-butyl 4-ethyl 3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 6 as a slight yellow solid (3.7 g,yield: 46%). ESI-MS (M+H)⁺: 377.2.

Preparation of trans-1-tert-butyl 4-ethyl3-aminopiperidine-1,4-dicarboxylate. Trans-1-tert-butyl 4-ethyl3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate 6 (3.7 g, 8.3mmol, 1.0 equiv) was hydrogenated in the presence of 10% Pd/C (0.37 g)catalyst under H₂ at 30 atmospheric pressure in MeOH (100 mL) at 50° C.for 8 h. The catalyst was filtered off and the solvent was removed invacuo to give (trans)-1-tert-butyl 4-ethyl3-aminopiperidine-1,4-dicarboxylate 7 as light yellow oil (2.5 g, yield:92%). ESI-MS (M+H)⁺: 273.1. ¹H NMR (400 MHz, CDCl₃) δ: 4.18 (q, 2H),3.97-3.94 (m, 2H), 3.37 (s, 1H), 3.07-3.02 (m, 1H), 2.89-2.85 (m, 1H),2.60-2.55 (m, 1H), 2.01-1.91 (m, 1H), 1.70-1.54 (m, 3H), 1.46 (s, 9H),1.28 (t, 3H).

Synthesis of trans-1-tert-butyl 4-ethyl3-(5-bromopentanamido)piperidine-1,4-dicarboxylate. To a solution oftrans-1-tert-butyl 4-ethyl 3-aminopiperidine-1,4-dicarboxylate 7 (2.5 g,9.2 mmol, 1.0 equiv) in CH₂Cl₂ (50 mL), Et₃N (2.5 mL, 18.4 mmol, 2.0equiv) was added at rt. After the reaction solution was stirred at rtfor 10 min, 5-bromovaleryl chloride (1.9 g, 9.6 mmol, 1.05 eq) wasadded. The reaction solution was stirred at rt for 2 h. The mixture wasquenched with H₂O (20 mL) and extracted with CH₂Cl₂ (50 mL×3). Theorganic layer was collected, concentrated in vacuo, and the residue waspurified by column chromatography (silica, petroleum ether/EtOAc=1:1) togive (trans)-1-tert-butyl 4-ethyl3-(5-bromopentanamido)piperidine-1,4-dicarboxylate 8 as yellow oil (3.2g, yield: 80%). ESI-MS (M+H−56)⁺: 379.0. ¹H NMR (400 MHz, CDCl₃) δ: 5.99(d, 1H), 4.39-4.38 (m, 1H), 4.15 (q, 2H), 3.79-3.74 (m, 1H), 3.66-3.60(m, 1H), 3.41 (t, 2H), 3.30-3.26 (m, 1H), 3.21-3.14 (m, 1H), 2.78-2.74(m, 1H), 2.19 (t, 2H), 1.99-1.85 (m, 3H), 1.80-1.72 (m, 3H), 1.45 (s,9H), 1.27 (t, 3H).

Synthesis of trans-1′-tert-butyl 4′-ethyl2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate. To a solution oftrans-1-tert-butyl 4-ethyl3-(5-bromopentanamido)piperidine-1,4-dicarboxylate 8 (3.0 g, 6.9 mmol,1.0 equiv) in THE (20 mL), NaH (276 mg, 6.9 mmol, 1.0 equiv) wascarefully added in small portions at 0° C. The reaction solution wasstirred at reflux condition for 4 h. The mixture was quenched with H₂O(20 mL), and extracted with EtOAc (30 mL×3). The organic layer wascollected, dried (Na₂SO₄), filtered, and concentrated in vacuo. Theresidue was purified by column chromatography (silica, petroleumether/EtOAc=1:2) to give (trans)-1′-tert-butyl 4′-ethyl2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate 9 as a slight yellow oil(2.1 g, yield: 88%). ESI-MS (M+H−56): 299.1. ¹H NMR (400 MHz, CDCl₃) δ:4.10 (q, 4H), 3.38-3.19 (m, 4H), 2.70-2.61 (m, 1H), 2.36-2.31 (m, 2H),1.95-1.92 (m, 1H), 1.75-1.71 (m, 6H), 1.46 (s, 9H), 1.23 (t, 3H).

Example 2 Preparation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide

Synthesis oftrans-1′-tert-butyl-4′-ethyl-3-iodo-2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate.To the solution of trans-1′-tert-butyl 4′-ethyl2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate 8 (141 mg, 2.58 mmol, 1.0equiv) in dry toluene (10 mL) at 0° C., TMEDA (0.89 g, 7.7 mmol, 3.0equiv) and TMSCl (0.6 mg, 1.0 mmol, 2.0 equiv) were added successivelyunder N₂. After 0.5 h, 12 (0.98 g, 3.87 mmol, 1.5 equiv) was carefullyadded in small portions. The reaction solution was stirred at 0° C. tort for 16 h. The mixture was diluted with EtOAc (100 mL), washed withsaturated Na₂S₂O₃ (20 mL×2) and brine (20 mL), dried (Na₂SO₄), filtered,and concentrated in vacuo. The crude product 9 (2.2 g, Y: 81%) was useddirectly in the next step without further purification. ESI-MS(M+H−56)⁺: 424.9. ¹H NMR (400 MHz, CDCl₃): 4.78-4.73 (m, 1H), 4.19-4.04(m, 4H), 3.55-3.30 (m, 4H), 3.24-3.16 (m, 2H), 2.73-2.60 (m, 1H),2.22-2.14 (m, 2H), 1.96-1.78 (m, 2H), 1.70-1.60 (m, 1H), 1.44 (s, 9H),1.25 (t, J=7.2 Hz, 3H).

Synthesis of trans-1′-tert-butyl 4′-ethyl3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate. A 1.0 Msolution of lithium bis(trimethyldisilyl)amide in THE (13 mL, 12 mmol,2.0 equiv) was added through an addition funnel at 10-15° C. to asolution of 3-chloro-5-(trifluoromethyl)aniline (15 g, 78 mmol, 1.2equiv) in THE (13 mL). The mixture was allowed to stir at roomtemperature for 20 min and a solution of crudetrans-1′-tert-butyl-4′-ethyl-3-iodo-2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate9 (3.7 g, 65 mmol, 1.0 equiv) in THE (13 mL) was added through anaddition funnel at 10-15° C. over 30 min. After addition, the reactionwas allowed to stir at the temperature for 30 min. Upon completion, thereaction was cooled to 5° C. and quenched slowly with water (10 mL),keeping the temperature below 20° C. The quenched reaction was extractedwith EtOAc (2×30 mL). The combined organic layers were washed withsaturated brine (30 mL), dried (Na₂SO₄), filtered, and concentrated invacuo. The resulting crude product was purified over silica gel elutingwith a gradient of 10% to 75% of EtOAc in heptanes to give the desireproduct 10. ESI-MS (M+H−56)⁺: 463.1. ¹H NMR (400 MHz, CDCl₃) δ: 6.92 (s,1H), 6.71-6.69 (m, 2H), 4.17-4.06 (m, 4H), 3.78-3.68 (m, 2H), 3.46-3.36(m, 3H), 3.23-3.07 (m, 2H), 2.73-2.65 (m, 1H), 2.44-2.37 (m, 1H),2.03-1.85 (m, 3H), 1.71-1.61 (m, 2H), 1.46 (s, 9H), 1.27-1.19 (m, 3H).

Synthesis oftrans-1′-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl) phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylic acid. To a solution oftrans-1′-tert-butyl 4′-ethyl3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-1′,4′-dicarboxylate10 (180 mg, 0.33 mmol, 1.0 equiv) in EtOH (5 mL) was added NaOH (40 mg,0.99 mmol, 3.0 equiv) and solution was stirred at 80° C. for 1 h. Thesolvent was concentrated in vacuo and the residue was suspended in water(10 mL) and adjusted to pH=6 with HCl (4 N). The precipitate wasfiltered to afford(trans)-1′-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid 11 (150 mg, Y: 82%) as yellow solid which was used next stepwithout further purification. ESI-MS (M+H−85)⁺: 463.1. ¹H NMR (400 MHz,CDCl₃) δ: 6.85 (s, 1H), 6.82 (s, 1H), 6.78 (s, 1H), 4.12-3.96 (m, 4H),3.53-3.37 (m, 2H), 3.11-3.04 (m, 2H), 2.75-2.67 (m, 1H), 2.24-2.18 (m,1H), 1.98-1.89 (m, 3H), 1.71-1.58 (m, 2H), 1.44 (s, 9H).

Synthesis of trans-tert-butyl4′-carbamoyl-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-1′-carboxylate. To the solutionof trans1′-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid 11 (70 mg, 0.14 mmol, 1.0 equiv) in DMF (2 mL), was added NH₄Cl (22mg, 0.41 mmol, 3.0 equiv), HBTU (103 mg, 0.270 mmol, 2.0 equiv) andDIPEA (52 mg, 0.41 mmol, 3.0 equiv). The reaction solution was stirredat rt for 16 h, diluted with EtOAc (10 mL) and washed with water (5 mL)and brine (5 mL). The organic phase was separated and concentrated invacuo to afford a crude oil which was purified by pre-HPLC (MeOH/H₂Owith 0.05% TFA as mobile phase) to give the compound(trans)-tert-butyl4′-carbamoyl-3-((3-chloro-5-(trifluoromethyl) phenyl)amino)-2-oxo-[1,3′-bipiperidine]-1′-carboxylate 12 (60 mg, yield: 86%)as a light solid. ESI-MS (M+H−56)⁺: 463.1. ¹H NMR (400 MHz, CD₃OD) δ:6.87-6.86 (m, 1H), 6.84-6.83 (m, 1H), 6.80 (s, 1H), 4.11-4.03 (m, 3H),3.53-3.35 (m, 2H), 3.20-3.08 (m, 2H), 2.77-2.74 (m, 1H), 2.25-2.18 (m,1H), 1.99-1.88 (m, 3H), 1.70-1.60 (m, 2H), 1.46 (s, 9H).

Synthesis oftrans-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.To the solution of trans-tert-butyl4′-carbamoyl-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-1′-carboxylate12 (60 mg, 0.11 mmol) in CH₂Cl₂ (1.0 mL) was added CF₃CO₂H (1.0 mL) atrt. The reaction mixture was stirred at rt for 2 h, concentrated invacuo to give desired product 13 (43 mg, 90%) which was used directly inthe next step without further purification. ESI-MS (M+H)⁺: 419.0.

Synthesis oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.To a solution oftrans-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide13 (42 mg, 0.10 mmol, 1.0 equiv) in 1-butanol (2 mL),6-chloro-5-fluoropyrimidin-4-amine (18 mg, 0.12 mmol, 1.2 equiv) wasadded DIPEA (26 mg, 0.20 mmol, 2.0 equiv). The reaction solution wasstirred at 120° C. for 16 h. The mixture was diluted with EtOAc (20 mL),washed with H₂O (10 mL) and brine (10 mL), dried (Na₂SO₄), filtered, andconcentrated in vacuo. The crude was by purified by pre-HPLC (MeOH/H₂Owith 0.05% TFA as mobile phase) to give the compound(trans)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 (44 mg, yield: 83%) as a yellow solid. ESI-MS (M+H)⁺: 530.0. HPLC:(214 nm: 100%, 254 nm: 100%). ¹H NMR (400 MHz, CD₃OD) δ: 7.97 (s, 1H),6.84 (s, 1H), 6.81 (s, 1H), 6.76 (s, 1H), 4.58-4.52 (m, 2H), 4.09-4.03(m, 1H), 3.52-3.35 (m, 3H), 3.29-3.27 (m, 4H), 3.12-3.05 (m, 1H),2.24-2.17 (m, 1H), 2.02-1.91 (m, 3H), 1.80-1.63 (m, 2H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide. The mixture offour diastereomers of compound 14 was separated into three peaks by SFC(IA(2×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 60 ml/min) and the titlecompound corresponded to peak 3. LCMS (Agilent460, 254 nm): ES (+) MSm/e=530.1 (M+1) @ 1.20 min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J=2.01Hz, 1H), 7.38 (br. s., 1H), 6.94 (s, 2H), 6.75-6.87 (m, 2H), 6.41-6.66(m, 3H), 4.29 (br. s., 1H), 4.23 (d, J=13.05 Hz, 1H), 3.96-4.18 (m, 2H),3.44 (td, J=6.15, 12.30 Hz, 1H), 3.24-3.33 (m, 1H), 3.10 (br. s., 1H),2.88 (br. s., 1H), 2.82 (t, J=12.30 Hz, 1H), 2.13 (qd, J=5.94, 12.30 Hz,1H), 1.74-1.93 (m, 3H), 1.58-1.74 (m, 1H), 1.41-1.58 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers of compound 14 was separated intothree peaks by SFC (IA(2×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 60ml/min) and the title compound corresponded to peak 2. LCMS (Agilent460,254 nm): ES (+) MS m/e=530.1 (M+1) @ 1.19 min. ¹H NMR (400 MHz, DMSO-d6)δ: 7.77 (d, J=1.76 Hz, 1H), 7.39 (br. s., 1H), 6.98 (s, 1H), 6.96 (s,1H), 6.72-6.88 (m, 2H), 6.57 (s, 2H), 6.54 (d, J=7.78 Hz, 1H), 4.05-4.33(m, 4H), 3.37 (t, J=6.27 Hz, 2H), 3.11 (br. s., 1H), 2.94 (br. s., 1H),2.82 (t, J=12.30 Hz, 1H), 2.02-2.16 (m, 1H), 1.75-1.92 (m, 3H),1.57-1.74 (m, 1H), 1.36-1.54 (m, 1H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers of compound 14 was separated intothree peaks by SFC (IA(2×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 60ml/min). Peak 1 of 3 was further purified SFC (AD-H (2×15 cm), 30% iPrOH(0.1% DEA)/CO₂, 100 bar, 60 ml/min) to afford the title compound. LCMS(Agilent 460, 254 nm): ES (+) MS m/e=530.1 (M+1) @ 1.20 min. ¹H NMR (400MHz, DMSO-d6) δ: 7.77 (d, J=1.76 Hz, 1H), 7.38 (br. s., 1H), 6.94 (s,2H), 6.83 (s, 1H), 6.80 (s, 1H), 6.42-6.66 (m, 3H), 4.18-4.47 (m, 2H),3.95-4.18 (m, 2H), 3.39-3.52 (m, 1H), 3.24-3.31 (m, 1H), 3.10 (br. s.,1H), 2.88 (br. s., 1H), 2.82 (t, J=12.30 Hz, 1H), 2.13 (qd, J=5.91,12.39 Hz, 1H), 1.73-1.92 (m, 3H), 1.58-1.73 (m, 1H), 1.42-1.58 (m, 1H).

(3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers of compound 14 was separated intothree peaks by SFC (IA(2×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 60ml/min). Peak 1 of 3 was further purified SFC (AD-H (2×15 cm), 30% iPrOH(0.1% DEA)/CO₂, 100 bar, 60 ml/min) to afford the titled compound. LCMS(Agilent 460, 254 nm): ES (+) MS m/e=530.1 (M+1) @ 1.20 min. ¹H NMR (400MHz, DMSO-d6) δ: 7.77 (d, J=1.76 Hz, 1H), 7.39 (br. s., 1H), 6.98 (s,1H), 6.96 (s, 1H), 6.73-6.88 (m, 2H), 6.57 (s, 2H), 6.54 (d, J=7.78 Hz,1H), 4.05-4.35 (m, 4H), 3.37 (t, J=6.15 Hz, 2H), 3.12 (br. s., 1H), 2.94(br. s., 1H), 2.82 (t, J=12.30 Hz, 1H), 2.09 (sxt, J=5.80 Hz, 1H),1.74-1.92 (m, 3H), 1.56-1.73 (m, 1H), 1.36-1.52 (m, 1H).

Example 3 Alternative Synthesis of(3R,3′R,4's)-1′-(6-Amino-5-Fluoropyrimidin-4-Yl)-3-((3-Chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide

In addition to the methods described in Example 2,(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide (compound I-1)was also synthesized according to Scheme 6.

1-tert-Butyl 4-ethyl 3-oxopiperidine-1,4-dicarboxylate 3-2. To asolution of 3-1 (5.0 kg, 19.1 mol, 1.0 equiv) in EtOH (50 L) under N₂was added (Boc)₂O (4.2 kg, 19.1 mol, 1.0 equiv), Et₃N (1.9 kg, 19.1 mol,1.0 equiv) and 10% Pd(OH)₂/C (250 g, 10% w/w). After evacuated andrefilled with hydrogenation three times, the mixture was stirred under 1atm of hydrogen at 50° C. for 15 hr. LC-MS indicated completelyconsumption of 3-1. After the mixture was cooled to ambient temperature,the catalyst was filtered through a layer of celite and washed with EtOH(2.5 L). The filtrate was concentrated in vacuo to afford crude 3-2 (5.2kg) as an oil, which was used in next step without further purification.

(S)-1-tert-butyl 4-ethyl3-((1-phenylethyl)amino)-5,6-dihydropyridine-1,4 (2H)-dicarboxylate(3-3). To a 100 L reactor equipped with Dean-Stark apparatus was chargedtoluene (20 L), crude compound 3-2 (5.2 kg, 19.1 mol, 1.0 equiv) rinsedwith toluene (30 L), pTSA (329 g, 0.2 mol, 0.01 equiv), andS-(−)-α-methylbenzylamine 0.95 kg, 16.2 mol, 0.85 equiv). The mixturewas heated to reflux with a nitrogen blanket and the water was removedthrough Dean-Stark. After 18 hours, LC-MS indicated complete consumptionof 3-2. The mixture was then cooled to the ambient temperature. Theinsolubles were removed by filtration, and the filtrate was concentratedin vacuo to dryness to afford crude 3-3 as a thick oil. This crudeproduct was used in the next step without further purification. 3-10% ofamide byproduct formed in this reaction and its structure wastentatively assigned based on LC-MS data.

(3R)-1-tert-butyl 4-ethyl3-(((S)-1-phenylethyl)amino)piperidine-1,4-dicarboxylate (3-4). To a 100L reactor charged with NaBH₄ (1.16 kg, 30.5 mol, 2.0 equiv) andanhydrous THE (60 L) under nitrogen was added TFA (10.5 kg, 92 mol, 6.0equiv) slowly over 30 min while maintaining temperature at 0-5° C. Themixture was then cooled to −45° C. In a separation reactor, crudeproduct 3-3 was dissolved in anhydrous acetonitrile (30 L), which wasadded slowly to the above solution of NaBH₄/TFA while maintaining theinternal temperature between −45˜−30° C. The mixture was stirred at −45°C. for 1 h, after which time, HPLC indicated complete consumption ofcompound 3-3. The mixture was slowly diluted with ice water (50 kg) andthe mixture was then warmed to 10° C. The product was extracted withEtOAc (2×40 L) and the combined organic layers were washed withsaturated NaHCO₃ solution (20 L). pH of the aqueous was ˜8. The organiclayers were dried (Na₂SO₄) and concentrated in vacuo to nearly drynessto afford a residue which was further azeotroped with MeOH (10 L×3) toremove excess EtOAc. In the end, a 10 L solution of crude 3-4 in MeOHwas obtained, which was used directly in the subsequent step withoutfurther purification. ESI-MS (M+H−1)⁺: 377.2. ¹H NMR (400 MHz, CD₃OD) δ:7.31-7.22 (m, 5H), 4.20 (q, 2H), 4.11-3.86 (m, 3H), 3.15 (s, 1H),3.00-2.90 (m, 2H), 2.64 (d, 2H), 1.87-1.85 (m, 1H), 1.68 (s, 1H),1.50-1.25 (m, 15H).

(3R)-1-(tert-butoxycarbonyl)-3-(((S)-1-phenylethyl)amino)piperidine-4-carboxylicacid (3-5). To a 100 L reactor were charged a with THF/MeOH (1:1, 80 L),was added a solution of LiOH H₂O (2.5 kg, 60 mol, 4.0 equiv) in water(10 L) and a solution of crude 3-4 in MeOH (10 L) from the above step.The resulting mixture was stirred at 22° C. for 18 hours, at which timeLC/MS indicated complete consumption of starting material 3-4. Thesolution was diluted with MTBE (40 L) and stirred for 20 min. Theaqueous layer was separated, cooled to 0° C. and neutralized with 3N HClsolution to pH between 7-8, while maintaining the internal temperaturebelow 10° C. The solution was washed with DCM (5×30 L) or until theLC/MS indicated no product 3-5 remained in the aqueous layer. Thecombined organic layers were concentrated in vacuo to dryness, suspendedin EtOAc and petroleum ether (2:1, 10 L) and stirred for 2 hours, thesolids were filtered, washed by petroleum ether (5 L) and dried undervacuum at 50° C. for 18 hours to give product (3.5 Kg, 53% yield) as asolid with 95% purity. Compound 3-5 is a mixture of ˜30:70 trans/cis atC-4 and ˜93:7 R:S at C-3. The average overall yield from 3-1 is 43-55%.ESI-MS (M+H−1)⁺: 349.2. ¹H NMR (400 MHz, CD₃OD) δ: 8.22-8.06 (m, 5H)4.11 (m, 1H), 3.86-3.82 (m, 1H), 3.59-3.56 (m, 1H), 2.79-2.65 (m, 1H),3.22-2.62 (m, 2H), 2.06-2.16 (m, 12H).

(3R)-1-(6-amino-5-fluoropyrimidin-4-yl)-3-(((S)-1-phenylethyl)amino)piperidine-4-carboxylicacid (3-7). To a 50 L reactor was charged with 10 L of 2N HCl and 3-5(850 g, 2.44 mol, 1.0 equiv). The mixture was warmed to 30° C. andstirred for 2 hours, at which time HPLC indicted complete consumption ofstarting 3-5. The solution was diluted with MTBE (4 L) and stirred for20 min, layers were separated and to the aqueous layer was added solidK₂CO₃ (660 g) over 1 hour to pH ˜7. Additional K₂CO₃ (660 g, 4.8 mol,2.0 equiv) was added following by 6-chloro-5-fluoropyrimidine-4-ylamine(360 g, 2.44 mole 1.0 equiv) and 1,4-dioxane (5 L). The mixture washeated to gentle reflux at 100° C. and stirred at this temperature for16 hours. HPLC indicated <2% of compound 3-6 remained. The mixture waswashed with DCM (2×5 L) and the organic wash solutions were discarded.The aqueous layer was treated with active carbon (425 g) by stirring theslurry for 1 hour at 30° C. followed by filtration through diatomite.This active carbon treatment was repeated. The resulting aqueoussolution was neutralized to pH ˜7 with concentrated HCl, and stirred at22° C. for 3 hours, the resulting slurry was filtered and the wet cakewas washed with washed with 1,4-dioxane/water (1:1, 1.2 L), dried undervacuum at 50° C. for 18 hr until KF ˜0.5%. of product 3-7 was obtainedas a pale white solid (690 g, 81% yield) with purity of 98.6%. Theproduct contains a mixture of 1:9 cis/trans iomers at the C3 and C4positions. ESI-MS (M+H−1)⁺: 460.2. ¹H NMR (400 MHz, CD₃OD) δ: 8.49 (d,J=2.01 Hz, 1H), 8.21-8.14 (m, 5H) 4.94-4.90 (m, 1H), 4.63 (d, J=11.55Hz, 1H), 4.42 (m, 1H), 4.03 (m, 2H), 3.59-3.72 (m, 3H), 2.84-2.93 (m,1H), 2.20-2.31 (m, 1H), 2.15 (d, J=6.78 Hz, 3H).

(3R)-3-amino-1-(6-amino-5-fluoropyrimidin-4-yl)piperidine-4-carboxylicacid (3-8). To a 10 L reaction were charged under N₂, i-PrOH (3.5 L),H₂O (3.5 L), 3-7 (1.0 equiv, 0.97 mol, 350 g), potassium fluoride monohydrate (290 g, 3.0 eq, 3.0 mol) and 35 g of 20% Pd(OH)₂/C (10% v/w).After evacuated/refilled with hydrogen three times, the mixture waswarmed to 40-50° C. and vigorously stirred at that temperature under 1atmosphere of hydrogen. After 18 hours, LC/MS indicated <1% of startingmaterial 3-7 remained. The mixture was purged with N₂ for 20 min, cooledto 22° C., and filtered. Both the wet cake and filtrate contained theproduct and processed separately.

The filtrate was concentrated in vacuo at 50° C. to a volume of −200 mL.After cooled to 20° C. and stirred at this temperature for 2 hours, aslurry was obtained, and the solid was filtered, washed with water (400mL) and dried under vacuum and at 50° C. to give product 3-8 (65 g). Thewet cake from the reaction filtration was stirred in 1 N HCl (1 L) for 2hours to dissolve the product and the remaining catalyst solid was thenremoved by filtration. The acidic filtrate was neutralized with solidLiOH to pH ˜7 to precipitate the product 3-8. The product was washedwith water (200 mL), dried under vacuum and at 50° C. to give 120 g ofproduct. A total of 185 g of product was obtained with 98.7% purity andin 75% yield based on H NMR. All the mother liquors were combined andconcentrated to a volume of ˜400 mL result in a slurry, filtration, washwith water and drying gave additional 64 g solid with 50% purity. ¹H NMR(400 MHz, D20): δ 7.77 (s, 1H) 4.12 (d, J=14.05 Hz, 1H), 4.01 (d,J=13.05 Hz, 1H), 3.26 (d, J=13.80 Hz, 1), 2.99-3.10 (m, 1H), 2.64-2.73(m, 1H), 1.98 (dd, J=3.39, 14.18 Hz, 1H), 1.74-1.87 (m, 1H).

((3R,3′R)-1′-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid (3-10). To a solution of 3-8 (440 g, 1.9 mole, 1.0 equiv) in DMSO(10 L) was added 3-8 A sequentially (640 g, 1.9 mole, 1.0 equiv) andsodium triacetoxyborohydride (STAB, 402.0 g, 3.8 mole, 2 equiv) and Et₃N(190 g, 1.9 mol, 1.0 equiv). The mixture was heated to 50° C. andstirred for 3 hours to show complete conversion by HPLC to intermediate3-9.

The solution was diluted with MeOH (182 g, 5.7 mol, 3.0 eq) to quenchthe excess of STAB, and the reaction was heated to 70˜80° C. After 16hours, HPLC indicated 22% of product 3-10 formed and 61% intermediate3-9 remained and chiral HPLC indicated 3% lactam epimer. The mixture washeld at 70-80° C. for additional 24 hours to give 50% 3-10, 35% 3-9, and7% lactam epimer. After another 40 hours stirring, 80% 3-10 formed, 4%3-9 remained, and the lactam epimer increased to 14%. The mixture wascooled to 22° C., and quenched with 2N NH₄Cl solution (5 L) to give aslurry mixture. After 30 minute stirring, the mixture was filtered andthe wet cake was washed with water (3 L), dried under vacuum and at 55°C. until KF<0.1. Crude 3-10 was obtained as a brown solid (850 g,97.7%); chiral HPLC indicted 12.5% lactam epimer. This product was useddirectly without further purification

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid (3-10-trans). To a 10 L reactor was charged under N₂ with 3-10 (850g, 1.9 mol, 1.0 equiv) in DMF (4.25 L, 5 v/w) to give a clear solution,was added 4-dimethylaminopyridine (DMAP 116 g, 0.95 mol, 0.5 equiv) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 36.5 g, 0.19 mol,0.1 equiv). After the mixture was stirred at 15 to 22° C. for about 1hour, additional EDCI (36.5 g, 0.19 mol, 0.1 equiv) was added andstirred for another 1 hour. HPLC indicated a 69:1 trans/cis mixture. Theproduct 3-10-trans was not isolated and was converted to compound I-1 inone-pot. ¹H NMR (300 MHz, DMSO d6): δ 1.47-1.55 (m, 1H), 1.63-1.68 (m,1H), 1.81-1.87 (m, 1H), 1.90-1.97 (m, 1H), 2.93-3.19 (m, 1H), 3.16-3.23(m, 1H), 3.33-3.45 (m, 2H) 4.07-4.33 (m, 3H), 6.80 (m, 1H), 6.94-6.98(m, 1H), 7.10-7.16 (m, 2H), 7.91 (s, 1H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide(I-1). To the above reaction mixture, was charged at 22° C., withO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 600 g, 1.9 mol, 1.0 equiv),N,N-diisopropylethylamine (DIPEA, 1.0 kg, 9.5 mol, 5.0 equiv) andfinally NH₄Cl (260 g, 5.7 mol, 3.0 equiv). The resulting mixture wasstirred at 15° C. for 1 hour, HPLC indicated complete consumption of3-10-trans, the mixture was poured into brine (25 L) and extracted withEtOAc (2×2 L). The combined organics were washed with brine (2×2 L) andconcentrated in vacuo below 45° C. to dryness to result in a crude I-1,which was purified by chromatograph with EtOAc/petroleum ether/MeOH(1:1:0 to 50:50:10) to give three fractions, which contained 316 g,98.8% chemical purity and 10.8% epimer, 160 g, 82.3% chemical purity and17.5% epimer and 180 g, 61% purity and 11.3% epimer, respectively. Theabove first two fractions were combined and further purified byprep-HPLC to give 200 g product with >99% purity and <1% epimer. ¹H NMR(400 MHz, DMSO d6): δ 1.48-1.53 (m, 1H), 1.66-1.69 (m, 1H), 1.77-1.79(m, 3H), 2.11-2.16 (m, 1H), 2.80-2.88 (m, 2H), 3.11 (s, 1H), 3.42-3.48(m, 1H), 4.0-4.25 (m, 4H), 6.58 (s, 3H), 6.80-6.85 (d, J=10.2, 2H), 6.95(s, 2H), 7.40 (s, 1H), 7.77 (s, 1H).

Example 4 Alternative Syntheses of(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide

In addition to the methods described in Examples 2 and 3,(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide (compound I-1)was also synthesized according to Scheme 6.

(3R)-3-amino-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid(4-L-6). To a 10 L reactor charged under nitrogen with compound 4-5 (100g, 0.287 mole), MeOH(6 L, 60 v/w) and 10 g 20% Pd(OH)₂/C. The reactorwas evacuated/refilled with hydrogen three times and the mixture waswarmed to 40-50° C. while stirring under 3 Mpa of hydrogen for 40 hours.LC/MS indicated complete consumption of starting material 4-5. Themixture was cooled to 22° C. and filtered, and the filtrate wasconcentrated in vacuo to dryness to afford a solid product. This crudeproduct was slurried in EtOH (500 mL) at 22° C. for 2 hours, filteredand dried under vacuum at 50° C. to afford a 85% yield of product 4-L-6(60 g, 0.245 mole) as a white solid.

(3R)-1-(tert-butoxycarbonyl)-3-(((R)-4-((3-chloro-5-(trifluoromethyl)phenyl)amino)-5-ethoxy-5-oxopentyl)amino)piperidine-4-carboxylicacid (4-L-8). To a solution of 4-L-6 (48.4 g, 0.197 mole) in DMSO (450mL) was added Et₃N (20.2 g, 0.199 mole, 1 equiv), 3-8 A (67.4 g, 0.199mole, 1 equiv) and sodium triacetoxyborohydride (STAB, 84.8 g, 0.40mole, 2.0 equiv). The mixture was heated to 50° C. over 30 min andstirred at that temperature for 3 hours. LC/MS indicated consumption ofmost of starting material 4-L-6 and formation of 4-L-8.

The reaction was quenched by adding EtOH (35 mL) and stirring at 50° C.for 30 min. The mixture was heated at 75-85° C. for 3 days. The mixturewas cooled to 18° C. and transferred slowly into water (6 L) whilevigorously stirring to afford a slurry. After 2 hours, the solids werefiltered and washed with water (3×3 L), dried under vacuum at 60-70° C.for 24 hours to give 4-L-9 (114 g) as a brown solid. The solid was useddirectly in the subsequent step.

(3R,3′R,4′S)-1′-(tert-butoxycarbonyl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid(4-L-9-trans). To a solution of crude 4-L-9 (100 g), in DMF (500 mL) wasadded 4-dimethylaminopyridine (11 g, 0.09 mole, 0.5 equiv) and stirredat 20° C. for 10 min. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (7.0g, 0.036 mole, 0.2 equiv) was added and the reaction was stirred 20° C.for 3 hours. HPLC indicated a ratio of 57:43 cis/trans mixture andadditional EDAC (3.5 g, 0.018 mole, 0.1 equiv) was added. After 5 hours,HPLC indicated complete conversion to 4-L-9-trans. The mixture wastransferred to water (2.25 L) slowly and the mixture was extracted withEtOAc (2×500 mL), and the organic layers were washed with brine (500 mL)and water (500 mL), concentrated in vacuo to dryness to give crude4-L-9-trans (100 g) as a brown solid. The crude was dissolved in EtOAc(135 mL) at 60° C. and then cooled to 20° C. over 1 hour followed byadding 50 mL petroleum ether. The mixture was aged for 2 hours. Thesolids were filtered and washed with 3:1 EtOAc/petroleum ether (50 mL),dried under vacuum at 50° C. for 16 hours to give 4-L-9-trans (23 g, 22%yield with 99% purity). ¹H NMR (400 MHz, DMSO-d6) δ 6.94 (s, 2H), 6.81(s, 1H), 6.54-6.61 (m, 1H), 3.99-4.08 (m, 1H), 3.42-3.38 (m, 2H),2.07-2.16 (m, 1H), 1.74-1.92 (m, 3H), 1.39 (s, 9H).

(3R,3′R,4′S)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid hydrochloride (4-L-10 trans). To a solution of 0.5N HCl in EtOAc(76 mL) was added 4-L-9 trans (20 g, 38 mmol) and heated at 20° C. for18 h to give a slurry. The solid was filtered, washed with EtOAc (5 mL)and dried under vacuum at 45° C. for 18 h to afford 4-L-10 as the HClsalt (17 g, 97% yield).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid (3-10-trans). A solution of 4-L-10 (2.0 g, 4.38 mole),6-chloro-5-fluoro-pyrimidin-4-ylamine (711 mg, 4.82 mmole, 1.1 equiv),DIPEA (1.52 mL, 8.77 mole, 2 eq.) in 40 mL nBuOH was heated to 130-140°C. for 72 h. The mixture was cooled to 22° C. and concentrated in vacuoto afford a residue which purified by column to give 3-10-trans (1.1 g,47%). A relatively minor amount of epimer(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid was also observed. Intermediate 3-10-trans can be converted tocompound I-1 via procedure described above.

Compound I-1 was also synthesized according to Scheme 8.

The synthesis of (R)-tert-butyl1-(3-chloro-5-(trifluoromethyl)phenyl)-5-oxopyrrolidine-2-carboxylate4-e was synthesized using a similar procedure as in Phillips, D. P.;Zhu, X. —F.; Lau, T. L.; Yang, K.; Liu, H. Tetrahedron Letters, 2009,50, 7293, whereby the (S)-methyl 5-oxopyrrolidine-2-carboxylate and1-chloro-4-iodobenzene were substituted for the (R)-tert-butyl5-oxopyrrolidine-2-carboxylate and1-chloro-3-iodo-5-(trifluoromethyl)benzene.

(2R)-tert-butyl1-(3-chloro-5-(trifluoromethyl)phenyl)-5-hydroxypyrrolidine-2-carboxylate.An anhydrous solution of 4-e (11 g, 30 mmol) in Me-THF (100 mL) wascooled to −35° C. under an atmosphere of nitrogen. A solution of DIABL-H(5.9 g, 42 mmol) in toluene (42 mL) was added dropwise while maintainingthe temperature at −35° C. The reaction was monitored by HPLC and uponcompletion a solution of 1N Rochell salt (100 mL) was added whilemaintaining the reaction temperature below 0° C. The organic phase wasseparated, washed with 1N Rochell salt (50 mL×3) and separated, dilutedwith Et₃N (4 mL), dried (Na₂SO₄) and concentrated in vacuo to afford 4-f(8.3 g) as an oil.

(3R)-1-(6-amino-5-fluoropyrimidin-4-yl)-3-(((R)-5-(tert-butoxy)-4-((3-chloro-5-(trifluoromethyl)phenyl)amino)-5-oxopentyl)amino)piperidine-4-carboxylic acid. A solutionof 4-f (37.4 g, 0.146 mmol) in DMF (700 mL) was treated with 3-8 (40.2g, 0.11 mmol), Et₃N (10.1 g, 0.1 mmol) STAB (42.4 g, 0.2 mmol) and themixture was heated to 55° C. for 5 h. The reaction was diluted withwater (2.5 L), extracted with EtOAc (500 mL×3), the organic phases werecombined and washed with brine, separated, dried (Na₂SO₄) andconcentrated in vacuo to afford 4-a (40.2 g) as solid which was usedwithout any additional purification.

(3R)-1-(6-amino-5-fluoropyrimidin-4-yl)-3-(((R)-4-carboxy-4-((3-chloro-5-(trifluoromethyl)phenyl)amino)butyl)amino)piperidine-4-carboxylicacid. To a solution of 5 N HCl (250 mL) was added t-butyl ester 4-a andthe suspension was heated to 55° C. for 5 h while the hydrolysis wasmonitored by HPLC. Upon complete formation of the product the water wasremoved in vacuo resulting in a solid 4-b which was dried under vacuumand used without any additional purification.

(3R,3′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxylicacid. To a solution of acid 4-b (55 g, 0.1 mol) in DMF (500 mL) wasadded a DIEA (64.5 g, 0.5 mol), CDI (32.5 g, 0.2 mol) at 0° C. Thesolution was stirred for 1.5 h at 0° C., diluted with water (3 L),adjusted to a pH 3 with HCl and extracted with EtOAc (2 L×3). Theorganic phase were combined, dried (Na₂SO₄) and concentrated in vacuo toafford 4-c (48 g).

The remaining steps to compound I-1 are completed via proceduresdescribed above.

Example 5 Synthesis of trans-tert-butyl3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(methylcarbamoyl)-2-oxo-[1,3′-bipiperidine]-1′-carboxylate

Synthesis of trans-tert-butyl 3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(methylcarbamoyl)-2-oxo-[1,3′-bipiperidine]-1′-carboxylate. Asimilar procedure was used as described for the synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford the crude material which was purified by pre-HPLC (MeOH/H₂Owith 0.05% NH₃.H₂O as mobile phase) to give the title compound (360 mg,yield: 67%) as a yellow solid. ESI-MS (M+H)⁺: 544.18. HPLC: (214 nm:100.0%, 254 nm: 100.0%). ¹H NMR (400 MHz, CD₃OD) (mixture of isomers) δ:7.69-7.68 (m, 1H), 6.78 (s, 1H), 6.75 (s, 1H), 6.71 (s, 1H), 4.39-4.36(m, 2H), 4.09-4.03 (m, 1H), 3.53-3.31 (m, 3H), 3.20-3.10 (m, 1H),2.99-2.92 (m, 1H), 2.55 (s, 3H), 2.28-2.19 (m, 1H), 1.96-1.77 (m, 5H),1.68-1.58 (m, 1H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N-methyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into two peaks by SFC(AD-H (2×15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/C₂, 100 bar, 60ml/min). Peak 2 was further purified by SFC separation (AD-H (2×15 cm),30% iPrOH (0.15% DEA)/CO₂, 100 bar, 60 ml/min) to afford the titlecompound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e=544.1 (M+1) @ 1.24min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.72-7.85 (m, 2H), 6.92 (s, 2H), 6.81(s, 1H), 6.43-6.64 (m, 3H), 4.34 (br. s., 1H), 4.23 (d, J=13.05 Hz, 1H),3.93-4.19 (m, 2H), 3.37-3.49 (m, 1H), 3.22-3.30 (m, 1H), 3.13 (br. s.,1H), 2.84 (t, J=12.05 Hz, 2H), 2.57 (d, J=4.52 Hz, 3H), 2.13 (qd,J=6.05, 12.45 Hz, 1H), 1.60-1.89 (m, 4H), 1.40-1.58 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N-methyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into two peaks by SFC(AD-H (2×15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/C₂, 100 bar, 60ml/min). Peak 2 was further purified by SFC separation (AD-H (2×15 cm),30% iPrOH (0.15% DEA)/CO₂, 100 bar, 60 ml/min) to afford the titlecompound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e=544.1 (M+1) @ 1.24min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.83 (q, J=4.60 Hz, 1H), 7.77 (d,J=1.76 Hz, 1H), 6.97 (d, J=6.78 Hz, 2H), 6.81 (s, 1H), 6.58 (s, 2H),6.53 (d, J=7.78 Hz, 1H), 4.23 (d, J=13.05 Hz, 2H), 3.90-4.19 (m, 2H),3.14 (br. s., 1H), 2.92 (br. s., 1H), 2.74-2.90 (m, 1H), 2.55 (d, J=4.52Hz, 3H), 2.00-2.18 (m, 1H), 1.74-1.89 (m, 3H), 1.56-1.74 (m, 1H),1.34-1.50 (m, 1H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N-methyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into two peaks by SFC(AD-H (2×15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO₂, 100 bar, 60ml/min). Peak 1 was further purified by SFC (AD-H (2×15 cm), 30% MeOH(0.15% DEA)/CO₂, 100 bar, 60 ml/min) to afford the titled compound. LCMS(Agilent 460, 254 nm): ES (+) MS m/e=544.1 (M+1) @ 1.23 min. ¹H NMR (400MHz, DMSO-d6) δ: 7.72-7.85 (m, 2H), 6.92 (s, 2H), 6.81 (s, 1H), 6.57 (s,2H), 6.54 (d, J=7.53 Hz, 1H), 4.23 (d, J=13.30 Hz, 1H), 4.15 (dd,J=3.26, 12.30 Hz, 1H), 4.08 (td, J=7.06, 10.48 Hz, 1H), 3.36-3.47 (m,1H), 3.23-3.30 (m, 1H), 3.13 (br. s., 1H), 2.84 (t, J=11.80 Hz, 2H),2.57 (d, J=4.52 Hz, 3H), 2.13 (qd, J=6.17, 12.61 Hz, 1H), 1.62-1.91 (m,4H), 1.42-1.57 (m, 1H).

trans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N-methyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into two peaks by SFC(AD-H (2×15 cm), 50% 1:1 IPA:methanol (0.1% DEA)/CO₂, 100 bar, 60ml/min). Peak 1 was further purified by SFC (AD-H (2×15 cm), 30% MeOH(0.15% DEA)/CO₂, 100 bar, 60 ml/min) to afford the titled cmpd. LCMS(Agilent 460, 254 nm): ES (+) MS m/e=544.1 (M+1) @ 1.23 min. LCMS(Agilent 460, 254 nm): ES (+) MS m/e=544.1 (M+1) @ 1.24 min. ¹H NMR (400MHz, DMSO-d6) δ: 7.80-7.89 (m, 1H), 7.72-7.80 (m, 1H), 6.97 (d, J=6.53Hz, 2H), 6.81 (s, 1H), 6.58 (s, 2H), 6.53 (d, J=7.78 Hz, 1H), 4.23 (d,J=13.05 Hz, 2H), 3.89-4.19 (m, 2H), 3.13 (br. s., 1H), 2.74-3.02 (m,J=12.42, 12.42 Hz, 2H), 2.55 (d, J=4.52 Hz, 3H), 2.08 (qd, J=5.97, 12.20Hz, 1H), 1.81 (td, J=6.24, 12.36 Hz, 3H), 1.56-1.74 (m, 1H), 1.33-1.51(m, 1H).

Example 6

Synthesis oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.A similar procedure was used as described for the synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford the crude material which was purified by pre-HPLC (MeOH/H₂Owith 0.05% TFA as mobile phase) to give the title compound (45 mg,yield: 90%) as a yellow solid. ESI-MS (M+H)⁺: 558.0. HPLC: (214 nm:98.2%, 254 nm: 100.0%). ¹H NMR (400 MHz, CD₃OD) δ: 7.77 (s, 1H),6.92-6.89 (m, 1H), 6.83-6.81 (m, 1H), 6.79 (s, 1H), 4.38-4.38 (m, 2H),4.05-4.00 (m, 2H), 3.55-3.53 (m, 1H), 3.45-3.40 (m, 2H), 3.15-2.89 (m,7H), 2.21-2.16 (m, 1H), 1.90-1.86 (m, 3H), 1.66-1.56 (m, 2H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide using a twostep chiral SFC separation. Firstly, the mixture was separated into twopeaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H)⁺: 558.0 ¹H NMR (400 MHz, CDCl₃)δ: 7.92 (d, J=1.76 Hz, 1H), 6.94 (s, 1H), 6.72 (d, J=7.78 Hz, 2H), 5.21(d, J=3.51 Hz, 1H), 4.70 (s, 2H), 4.45 (dd, J=2.76, 12.80 Hz, 2H),4.17-4.32 (m, 1H), 3.64-3.80 (m, 2H), 3.44-3.58 (s, 3H), 3.09 (s, 3H),3.00-3.09 (m, 1H), 2.95 (s, 3H), 2.40 (dd, J=5.52, 13.30 Hz, 1H),1.63-1.99 (m, 3H), 1.26-1.43 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide using a twostep chiral SFC separation. Firstly, the mixture was separated into twopeaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H)⁺: 558.0 ¹H NMR (400 MHz, (400MHz, CDCl₃) δ: 7.93 (d, J=1.26 Hz, 1H), 6.93 (s, 1H), 6.69 (br. s., 2H),5.06 (d, J=4.27 Hz, 1H), 4.71 (s, 1H), 4.45 (d, J=12.55 Hz, 2H),4.16-4.26 (m, 1H), 3.66-3.81 (m, 2H), 3.54-3.64 (m, 1H), 3.40-3.54 (m,2H), 3.01-3.10 (m, 4H), 2.95 (s, 3H), 2.37 (dd, J=5.27, 13.05 Hz, 1H),1.91-2.02 (m, 2H), 1.48-1.75 (m, 2H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide using a twostep chiral SFC separation. Firstly, the mixture was separated into twopeaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H)⁺: 558.0 ¹H NMR (400 MHz, CDCl₃)δ: 7.92 (d, J=1.76 Hz, 1H), 6.94 (s, 1H), 6.72 (d, J=7.78 Hz, 2H), 5.21(d, J=3.51 Hz, 1H), 4.70 (s, 2H), 4.45 (dd, J=2.76, 12.80 Hz, 2H),4.19-4.30 (m, 1H), 3.66-3.79 (m, 2H), 3.47-3.56 (m, 3H), 3.09 (s, 3H),2.97-3.07 (m, 1H), 2.95 (s, 3H), 2.40 (dd, J=5.52, 13.30 Hz, 1H),1.81-1.97 (m, 3H), 1.73 (dd, J=3.76, 12.80 Hz, 1H).

(3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide using a twostep chiral SFC separation. Firstly, the mixture was separated into twopeaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and theneach mixture containing a pair of isomers was further separated into thesingle enantiomers using a ChiralPak IA (2×15 cm, 30% methanol w/0.1%DEA 100 bar) column. ESI-MS (M+H)⁺: 558.0 ¹H NMR (400 MHz, (400 MHz,CDCl₃) δ: 7.93 (d, J=1.26 Hz, 1H), 6.93 (s, 1H), 6.69 (br. s., 2H), 5.06(d, J=4.27 Hz, 1H), 4.71 (s, 1H), 4.45 (d, J=12.55 Hz, 2H), 4.16-4.26(m, 1H), 3.66-3.81 (m, 2H), 3.54-3.64 (m, 1H), 3.40-3.54 (m, 2H),3.01-3.10 (m, 4H), 2.95 (s, 3H), 2.37 (dd, J=5.27, 13.05 Hz, 1H),1.91-2.02 (m, 2H), 1.48-1.75 (m, 2H).

Example 7

Synthesis oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(4-methylpiperazine-1-carbonyl)-[1,3′-bipiperidin]-2-one.A similar procedure was used as described for the synthesis oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford 23 which was purified by reverse phase HPLC (MeOH/H₂O with0.05% NH₃.H₂O as mobile phase) to afford the title compound (100 mg,yield: 70%) as a yellow solid. ESI-MS (M+H)⁺: 613.24. ¹H NMR (400 MHz,CDCl₃) δ: 7.94 (s, 1H), 6.94 (s, 1H), 6.73-6.67 (m, 2H), 5.25-5.03 (m,1H), 4.71 (s, 2H), 4.49-4.40 (m, 2H), 4.35-4.16 (m, 1H), 3.82-3.64 (m,3H), 3.62-3.41 (m, 6H), 3.08-2.97 (m, 1H), 2.52-2.34 (m, 3H), 2.30-2.25(m, 2H), 2.20 (s, 3H), 1.85-1.64 (m, 2H), 1.72-1.64 (m, 2H), 1.49-1.31(m, 1H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(4-methylpiperazine-1-carbonyl)-[1,3′-bipiperidin]-2-one.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide 23 using atwo step chiral SFC separation. Firstly, the mixture was separated intotwo peaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H)⁺: 613.2 ¹H NMR (400 MHz, CD₃OD)δ. 7.95 (s, 1H), 6.83-6.97 (m, 3H), 4.50-4.68 (m, 2H), 4.28-4.40 (m,1H), 3.66-3.91 (m, 8H), 3.30-3.52 (m, 4H), 3.14 (t, J=12.42 Hz, 2H),2.73 (br. s., 2H), 2.27 (dd, J=5.65, 12.93 Hz, 1H), 1.83-2.05 (m, 2H),1.54-1.79 (m, 2H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(4-methylpiperazine-1-carbonyl)-[1,3′-bipiperidin]-2-one.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide 23 using atwo step chiral SFC separation. Firstly, the mixture was separated intotwo peaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H)⁺: 613.2. H NMR (400 MHz, CD₃OD)δ. 7.91-7.99 (m, 1H), 6.84-6.97 (m, 3H), 4.57 (dd, J=14.18, 19.70 Hz,2H), 4.34 (br. s., 1H), 3.42-3.53 (m, 1H), 3.37 (d, J=1.51 Hz, 3H),3.05-3.19 (m, 1H), 2.86 (t, J=7.53 Hz, 1H), 2.68-2.78 (m, 2H), 2.26 (dd,J=5.65, 12.93 Hz, 1H), 1.82-2.03 (m, 3H), 1.55-1.79 (m, 2H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(4-methylpiperazine-1-carbonyl)-[1,3′-bipiperidin]-2-one.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide 23 using atwo step chiral SFC separation. Firstly, the mixture was separated intotwo peaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and thenthe resulting mixture containing a pair of isomers was further separatedinto the single enantiomers using a ChiralPak IA (2×15 cm, 30% methanolw/0.1% DEA 100 bar) column. ESI-MS (M+H): 613.2. ¹H NMR (400 MHz, CD₃OD)δ: 7.94 (s, 1H), 6.87-6.93 (m, 3H), 4.57 (dd, J=14.18, 19.70 Hz, 1H),4.34 (br. s., 1H), 3.43-3.51 (m, 1H), 3.36-3.38 (m, 2H), 3.13 (t,J=12.30 Hz, 1H), 2.86 (t, J=7.53 Hz, 1H), 2.73 (br. s., 1H), 2.26 (dd,J=5.65, 12.93 Hz, 1H), 1.96-2.03 (m, 1H), 1.83-1.94 (m, 1H), 1.51-1.75(m, 1H).

((3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-4′-(4-methylpiperazine-1-carbonyl)-[1,3′-bipiperidin]-2-one.The title compound was obtained from chiral separation oftrans-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide 23 using atwo step chiral SFC separation. Firstly, the mixture was separated intotwo peaks containing a mixture of two diastereomers((3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamideand(3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-N,N-dimethyl-2-oxo-[1,3′-bipiperidine]-4′-carboxamide)using a ChiralPak IC (2×15 cm, 30% methanol w/0.1 DEA) column, and theneach mixture containing a pair of isomers was further separated into thesingle enantiomers using a ChiralPak IA (2×15 cm, 30% methanol w/0.1%DEA 100 bar) column. ¹H NMR (400 MHz, CD₃OD) δ: 7.96 (br. s., 1H), 6.90(br. s., 1H), 6.76 (d, J=10.04 Hz, 2H), 4.60 (t, J=14.06 Hz, 2H),4.19-4.32 (m, 1H), 3.67-3.78 (m, 1H), 3.43-3.54 (m, 3H), 3.35-3.38 (m,3H), 3.16 (t, J=12.42 Hz, 1H), 2.86 (t, J=7.40 Hz, 2H), 2.79 (s, 3H),2.32 (dd, J=5.02, 12.80 Hz, 1H), 1.89-2.07 (m, 4H), 1.62-1.76 (m, 5H),Example 8

The synthesis of1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.A similar procedure was used as described for the synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford the crude material which was purified by pre-HPLC (MeOH/H₂Owith 0.05% NH₃.H₂O as mobile phase) to give the title compound (175 mg,yield: 69%) as a yellow solid. ESI-MS (M+H)⁺: 514.19. HPLC: (214 nm:96.13%, 254 nm: 96.53%). ¹H NMR (400 MHz, CD₃OD) δ: 7.79-7.78 (m, 1H),6.76 (s, 1H), 6.63-6.54 (m, 2H), 4.41-4.36 (m, 2H), 4.08-4.06 (m, 1H),3.55-3.41 (m, 3H), 3.28-3.25 (m, 1H), 2.99-2.93 (m, 1H), 2.28-2.21 (m,1H), 1.98-1.78 (m, 6H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IA (3×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min) and peak 3corresponded to the titled compound. LCMS (Agilent 460, 254 nm): ES (+)MS m/e=514.0 (M+1) @ 1.09 min. ¹H NMR (400 MHz, CDCl₃) δ: 7.91 (br. s.,1H), 6.66 (d, J=8.53 Hz, 1H), 6.63 (s, 1H), 6.46 (d, J=10.79 Hz, 1H),6.12 (br. s., 1H), 5.47 (br. s., 1H), 5.16 (d, J=3.51 Hz, 1H), 4.91 (br.s., 2H), 4.35-4.54 (m, 2H), 3.82 (td, J=5.11, 10.60 Hz, 2H), 3.51-3.60(m, 1H), 3.34-3.48 (m, 3H), 2.96 (t, J=12.30 Hz, 1H), 2.35-2.47 (m, 1H),1.91-2.06 (m, 3H), 1.84 (dq, J=3.89, 12.76 Hz, 1H), 1.48-1.62 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IA (3×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min). Peak 2 of 3corresponded to desired compound. LCMS (Agilent 460, 254 nm): ES (+) MSm/e=514.0 (M+1) @ 1.10 min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J=1.76Hz, 1H), 7.39 (s, 1H), 6.85 (s, 1H), 6.81 (s, 1H), 6.74 (d, J=12.30 Hz,1H), 6.47-6.66 (m, 4H), 4.23 (d, J=12.80 Hz, 2H), 3.90-4.18 (m, 2H),3.34-3.46 (m, 2H), 3.12 (br. s., 1H), 2.94 (br. s., 1H), 2.82 (t,J=12.42 Hz, 1H), 2.10 (qd, J=5.75, 12.11 Hz, 1H), 1.74-1.92 (m, 3H),1.56-1.72 (m, 1H), 1.37-1.52 (m, 1H).

(3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IA (3×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min). Peak 1 of 3was further purified by SFC (IA (3×15 cm), 30% iPrOH (0.1% DEA)/CO₂, 100bar, 70 ml/min) to afford the titled compound. LCMS (Agilent 460, 254nm): ES (+) MS m/e=514.0 (M+1) @ 1.10 min. ¹H NMR (400 MHz, DMSO-d6) δ:7.77 (d, 1.5 Hz, 1H), 7.39 (s., 1H), 6.85 (s, 1H), 6.81 (s., 1H), 6.74(d, J=12.30 Hz, 1H), 6.47-6.66 (m, 4H), 4.16-4.46 (m, 2H), 3.95-4.16 (m,2H), 3.34-3.48 (m, 2H), 3.12 (br. s., 1H), 2.87-3.01 (m, 2H), 2.82 (t,J=12.30 Hz, 1H), 2.10 (qd, J=5.75, 12.11 Hz, 1H), 1.74-1.92 (m, 3H),1.54-1.74 (m, 1H), 1.35-1.52 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-fluoro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IA (3×15 cm), 30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min). Peak 1 of 3was further purified by SFC (IA (3×15 cm), 30% iPrOH (0.1% DEA)/CO₂, 100bar, 70 ml/min) to afford the titled compound. LCMS (Agilent 460, 254nm): ES (+) MS m/e=514.0 (M+1) @ 1.10 min. ¹H NMR (400 MHz, DMSO-d6) δ.7.77 (d, J=1.76 Hz, 1H), 7.38 (br. s., 1H), 6.84 (s, 2H), 6.70 (d,J=12.30 Hz, 1H), 6.47-6.65 (m, 4H), 4.18-4.48 (m, 2H), 3.92-4.18 (m,2H), 3.38-3.49 (m, 1H), 3.20-3.30 (m, 1H), 3.11 (br. s., 1H), 2.88-2.99(m, 1H), 2.83 (t, J=12.30 Hz, 1H), 2.14 (qd, J=6.03, 12.52 Hz, 1H),1.74-1.92 (m, 3H), 1.59-1.74 (m, 1H), 1.41-1.58 (m, 1H).

Example 9

The synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide. A similar procedure wasused as described for the synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford the crude material which was purified by pre-HPLC (MeOH/H₂Owith 0.05% NH₃.H₂O as mobile phase) to give the title compound (141 mg,Y: 30%) as a white solid. ESI-MS (M+H)⁺: 479.9. HPLC: (214 nm: 100%, 254nm: 100%). ¹H NMR (400 MHz, DMSO d6) δ: 7.78-7.77 (m, 1H), 7.40-7.38 (m,1H), 6.86-6.82 (m, 1H), 6.62-6.55 (m, 3H), 6.45-6.37 (m, 3H), 4.26-3.94(m, 4H), 3.47-3.39 (m, 1H), 3.20-3.03 (m, 2H), 2.90-2.78 (m, 2H),2.18-2.04 (m, 1H), 1.86-1.34 (m, 5H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IC (2×15 cm), 25% MeOH (0.1% DEA)/CO₂, 100 bar, 60 ml/min) to affordthe title compound as peak 3 respectively. LCMS (Agilent 460, 254 nm):ES (+) MS m/e=480.0 (M+1) @ 1.01 min. ¹H NMR (400 MHz, CDCl₃) δ: 7.90(br. s., 1H), 6.44 (d, J=8.53 Hz, 1H), 6.40 (s, 1H), 6.30 (br. s., 1H),6.23 (d, J=11.04 Hz, 1H), 5.63 (br. s., 1H), 5.09 (br. s., 1H), 4.94(br. s., 2H), 4.45 (d, J=12.80 Hz, 2H), 3.68-3.95 (m, 2H), 3.49-3.56 (m,2H), 3.35-3.46 (m, 2H), 2.89-2.99 (m, 1H), 2.31-2.44 (m, 1H), 1.90-2.04(m, 3H), 1.76-1.89 (m, 1H), 1.49-1.61 (m, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IC (2×15 cm), 25% MeOH (0.1% DEA)/CO₂, 100 bar, 60 ml/min) to affordthe title compound as peak 1 respectively. LCMS (Agilent 460, 254 nm):ES (+) MS m/e=480.0 (M+1) @ 1.01 min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77(d, J=1.76 Hz, 1H), 7.39 (s., 1H), 6.81 (s, 1H), 6.57 (s, 3H), 6.46 (d,J=12.30 Hz, 1H), 6.39 (dd, J=1.76, 8.78 Hz, 1H), 6.34 (d, J=7.53 Hz,1H), 4.28 (br. s., 1H), 4.23 (d, J=13.05 Hz, 1H), 4.13 (dd, J=2.76,12.30 Hz, 1H), 4.03 (td, J=6.56, 10.98 Hz, 1H), 3.33-3.46 (m, 2H), 3.11(br. s., 1H), 2.94 (br. s., 1H), 2.82 (t, J=12.30 Hz, 1H), 2.09 (qd,J=5.75, 12.11 Hz, 1H), 1.72-1.92 (m, 3H), 1.56-1.72 (m, 1H), 1.29-1.50(m, 1H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IC (2×15 cm), 25% MeOH (0.1% DEA)/CO₂, 100 bar, 60 ml/min). Peak 2 of 3was further purified by SFC (IA (3×15 cm), 30% iPrOH (0.1% DEA)/CO₂, 100bar, 60 ml/min) to afford the title compound. LCMS (Agilent460, 254 nm):ES (+) MS m/e=480.0 (M+1) @ 1.01 min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77(d, J=2.01 Hz, 1H), 7.37 (br. s., 1H), 6.84 (s, 1H), 6.57 (s, 2H), 6.55(br. s., 1H), 6.30-6.48 (m, 3H), 4.28 (br. s., 1H), 4.23 (d, J=13.05 Hz,1H), 4.13 (dd, J=3.39, 12.67 Hz, 1H), 3.97 (td, J=6.84, 10.42 Hz, 1H),3.38-3.50 (m, 1H), 3.22-3.29 (m, 1H), 3.10 (br. s., 1H), 2.71-2.97 (m,1H), 2.83 (t, J=12.30 Hz, 1H), 2.13 (qd, J=6.13, 12.49 Hz, 1H),1.73-1.91 (m, 3H), 1.58-1.73 (m, 1H), 1.39-1.53 (m, 1H).

(3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was separated into three peaks by SFC(IC (2×15 cm), 25% MeOH (0.1% DEA)/CO₂, 100 bar, 60 ml/min). Peak 2 of 3was further purified by SFC (IA (3×15 cm), 30% iPrOH (0.1% DEA)/CO₂, 100bar, 60 ml/min) to afford the title compound. LCMS (Agilent460, 254 nm):ES (+) MS m/e=480.0 (M+1) @ 1.01 min. ¹H NMR (400 MHz, DMSO-d6) δ 7.77(d, J=1.76 Hz, 1H), 7.39 (s, 1H), 6.81 (s, 1H), 6.57 (s, 3H), 6.46 (td,J=2.01, 12.30 Hz, 1H), 6.39 (td, J=1.95, 8.66 Hz, 1H), 6.34 (d, J=7.53Hz, 1H), 4.18-4.48 (m, 2H), 4.09-4.18 (m, 1H), 3.79-4.09 (m, 1H),3.33-3.45 (m, 2H), 3.11 (br. s., 1H), 2.92 (br. s., 1H), 2.82 (t,J=12.42 Hz, 1H), 2.09 (qd, J=5.75, 12.11 Hz, 1H), 1.74-1.93 (m, 3H),1.51-1.74 (m, 1H), 1.32-1.51 (m, 1H).

Example 10

The synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide. A similarprocedure was used as described for the synthesis of(3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide14 to afford the crude material which was purified by pre-HPLC (MeOH/H₂Owith 0.05% NH₃.H₂O as mobile phase) to give the title compound (320 mg,yield: 44%) as a yellow solid. ESI-MS (M+H)⁺: 546.16. HPLC: (214 nm:98.4%, 254 nm: 98.0%). ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (s, 1H), 7.39(s, 1H), 6.84-6.81 (m, 1H), 6.75-6.72 (m, 1H), 6.62-6.57 (m, 3H),6.52-6.47 (m, 2H), 4.24-3.98 (m, 4H), 3.47-3.40 (m, 1H), 3.17-2.99 (m,2H), 2.86-2.79 (m, 2H), 2.15-1.99 (m, 1H), 1.86-1.60 (m, 4H), 1.48-1.39(m, 1H).

(3R,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was purified by SFC (AD-H (2×25 cm),30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min) to afford the titlecompound. LCMS (Agilent460, 254 nm): ES (+) MS m/e=546.0 (M+1) @ 1.20min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J=1.76 Hz, 1H), 7.39 (br. s.,1H), 6.81 (s, 1H), 6.75 (s, 1H), 6.62 (s, 1H), 6.57 (s, 2H), 6.38-6.52(m, 2H), 4.23 (d, J=13.05 Hz, 2H), 3.92-4.18 (m, 2H), 3.34-3.45 (m, 2H),3.11 (br. s., 1H), 2.93 (br. s., 1H), 2.82 (t, J=12.30 Hz, 1H), 2.08(qd, J=5.75, 12.11 Hz, 1H), 1.74-1.92 (m, 3H), 1.56-1.73 (m, 1H),1.36-1.50 (m, 1H).

(3S,3′S,4′R)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was purified by SFC (AD-H (2×25 cm),30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min) to afford the titlecompound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e=546.0 (M+1) @ 1.23min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J=2.01 Hz, 1H), 7.37 (br. s.,1H), 6.84 (s, 1H), 6.72 (s, 1H), 6.60 (s, 1H), 6.57 (s, 2H), 6.43-6.54(m, 2H), 4.23 (d, J=13.05 Hz, 1H), 4.22 (br. s., 1H), 4.13 (dd, J=3.26,12.30 Hz, 1H), 4.01 (td, J=6.81, 10.23 Hz, 1H), 3.44 (td, J=6.18, 12.49Hz, 1H), 3.20-3.29 (m, 1H), 3.11 (br. s., 1H), 2.88 (br. s., 1H), 2.83(t, J=12.30 Hz, 1H), 2.12 (qd, J=6.05, 12.46 Hz, 1H), 1.73-1.91 (m, 3H),1.59-1.73 (m, 1H), 1.48 (td, J=9.41, 19.33 Hz, 1H).

(3S,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was purified by SFC (AD-H (2×25 cm),30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min) to afford the titlecompound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e=546.0 (M+1) @ 1.22min. ¹H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, J=2.01 Hz, 1H), 7.38 (br. s.,1H), 6.81 (s, 1H), 6.75 (s, 1H), 6.62 (s, 1H), 6.57 (s, 2H), 6.42-6.51(m, 2H), 4.23 (d, J=12.80 Hz, 2H), 3.97-4.18 (m, 2H), 3.34-3.44 (m, 2H),3.10 (br. s., 1H), 2.93 (br. s., 1H), 2.82 (t, J=12.17 Hz, 1H),2.03-2.15 (m, 1H), 1.77-1.90 (m, 3H), 1.57-1.73 (m, 1H), 1.37-1.48 (m,1H).

(3R,3′R,4′S)-1′-(6-amino-5-fluoropyrimidin-4-yl)-3-((3-chloro-5-(trifluoromethoxy)phenyl)amino)-2-oxo-[1,3′-bipiperidine]-4′-carboxamide.The mixture of four diastereomers was purified by SFC (AD-H (2×25 cm),30% EtOH (0.1% DEA)/CO₂, 100 bar, 70 ml/min) to afford the titledcompound. LCMS (Agilent 460, 254 nm): ES (+) MS m/e=546.0 (M+1) @ 1.23min. ¹H NMR (400 MHz, CDCl₃) δ: 7.93 (s, 1H), 6.60 (s, 1H), 6.52 (s,1H), 6.35 (s, 1H), 5.85 (br. s., 1H), 5.32 (br. s., 1H), 4.97-5.15 (m,1H), 4.78 (br. s., 2H), 4.46 (d, J=13.05 Hz, 2H), 3.67-3.87 (m, 2H),3.34-3.60 (m, 4H), 2.99 (t, J=12.17 Hz, 1H), 2.34-2.49 (m, 1H),1.91-2.08 (m, 3H), 1.83 (dq, J=3.76, 12.72 Hz, 1H), 1.47-1.74 (m, 1H).

Example 11

In vitro BTK kinase assay: BTK-POLYGAT-LS ASSAY. The purpose of the BTKin vitro assay was to determine compound potency against BTK through themeasurement of IC₅₀. Compound inhibition was measured after monitoringthe amount of phosphorylation of a fluorescein-labeled polyGAT peptide(Invitrogen PV3611) in the presence of active BTK enzyme (Upstate14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black96 well plate (costar 3694). For a typical assay, a 24 uL aliquot of anATP/peptide master mix (final concentration; ATP 10 uM, polyGAT 100 nM)in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 200 uM Na₃PO₄, 5mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) was added to eachwell. Next, 1 uL of a 4-fold, 40× compound titration in 100% DMSOsolvent was added, followed by adding 15 uL of BTK enzyme mix in 1×kinase buffer (with a final concentration of 0.25 nM). The assay wasincubated for 30 minutes before being stopped with 28 uL of a 50 mM EDTAsolution. Aliquots (5 uL) of the kinase reaction were transferred to alow volume white 384 well plate (Corning 3674), and 5 uL of a 2×detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody,Invitrogen PV3552) was added. The plate was covered and incubated for 45minutes at room temperature. Time resolved fluorescence (TRF) onMolecular Devices M5 (332 nm excitation; 488 nm emission; 518 nmfluorescein emission) was measured. IC₅₀ values were calculated using afour parameter fit with 100% enzyme activity determined from the DMSOcontrol and 0% activity from the EDTA control.

Selected compounds of formula I were tested and found to be active inthe polyGAT assay. Compounds I-1, I-2, I-3, I-4, I-5 and I-7 gave IC₅₀values of 0.73 nM, 0.68 nM, 2.07 nM, 0.63 nM, 1.6 nM, and 1.2 nMrespectively. Compound I-6 has an IC₅₀ value less than 1 nM. Comparatorcompound I^(C), shown below, produced an IC₅₀ value of 2.0 nM.

Example 12 Study Protocol to Determine Activation of the PXR NuclearReceptor in Human DPX2 Cells

a) Protocol Summary: PXR has been shown to be a primary nuclear receptorthat mediates drug-induced expression of CYP3A4 (Bertilsson G, et al.;Proc Natl Acad Sci USA. 1998 Oct. 13; 95(21):12208-13). Based on thispathway of CYP3A4 induction, cell-based PXR reporter gene assay iscommonly used to screen new molecular entities (NMEs) in early drugdiscovery stage, for their potential to induce CYP3A4 (Luo G, et al.;Drug Metab Dispos. 2002 July; 30(7):795-804.) Studies were designed toevaluate the effect of new molecular entities (NMEs) on the activationof human PXR in DPX2 cells. Cell lines stably transfected with the PXRnuclear receptor and corresponding response elements were seeded into96-well plates. Twenty-four hr after seeding, cells were treated with 6distinct concentrations of NMEs in triplicate wells (see below), andcells then returned to the incubator for an additional 24 hr. At the endof this incubation period, the number of viable cells/well weredetermined using Promega's Cell Titer Fluor cytotoxicity assay.Following this assay, Promega's ONE-Glo was added to the same wells andreporter gene activity assessed.

b) Test System: The test system consisted of the stably transformed DPX2tumor cell line plated on 96-well microtiter plates. An expressionvector harboring the PXR nuclear receptor plus the appropriate enhancersand promoters linked to the luciferase reporter gene have been stablyintegrated into these tumor cell lines. Receptor activation was assessedby monitoring reporter gene activity, and by comparing the results tovehicle-treated cells. Positive controls consist of cells treated with 6different concentrations (0.1, 0.5, 1, 5, 10, and 20 μM) of rifampicin.In this manner, compounds activating PXR can be easily and rapidlyidentified. Since stably-integrated cell lines were used, it is possibleto observe from 3- to 70-fold receptor activation.

c) Data Processing and Receptor Activation Kinetics: Data processedusing MS-Excel was calculated as the mean (n=3) and % CV of the fold PXRactivation relative to vehicle-treated cells at each of the 6 differentdoses. All activation data was normalized to the number of viablecells/well. Results were also expressed as a percentage of the responsegiven by the appropriate positive control at a 10 M dose. EC₅₀ andE_(max) values were derived for test compounds that give receptoractivation using nonlinear regression of typical log dose-responsecurves (Prism V5.0c, GraphPad Software, San Diego, Calif.). Agentsexhibiting atypical dose-response curves were not analyzed in thisfashion.

d) New Molecular Entities (NMEs): Test compounds were tested at 0.05,0.1, 0.5, 1, 2.5, and 10 μM

Selected compounds of formula I were tested in the PXR assay. CompoundsI-1, I-2, I-3, I-4, and I-5 gave PXR % induction (relative to 10 uMrifampin) of 62%, 42%, 47%, 67%, and 90%, respectively. Comparatorcompound I^(C), shown above, produced a PXR % induction of 95%.

Example 13 Protocol for FastPatch hERG Inhibition Assay

The cardiac potassium channel, hERG, is responsible for a rapid delayedrectifier current (I_(Kr)) in human ventricle and inhibition of I_(Kr)is the most common cause of cardiac action potential prolongation bynon-cardiac drugs (see, e.g., Weirich and Antoni, Basic Res. Cardiol.,93, Suppl. 1, 125-32, 1998; Yap and Camm, Clin. Exp. Allergy, 29, Suppl.3, 174-81, 1999). Increased action potential duration has been cited asa factor in causing prolongation of the QT interval that has beenassociated with a dangerous ventricular arrhythmia, torsade de pointes(Brown and Rampe, Pharmaceutical News, 7, 15-20, 2000).

The in vitro effects of provided compounds was investigated on the hERG(human ether-à-go-go-related gene) potassium channel current (asurrogate for I_(Kr), the rapidly activating, delayed rectifier cardiacpotassium current) expressed in human embryonic kidney (HEK293) cellsstably transfected with hERG cDNA. Cells were placed in HEPES-bufferedphysiological saline solution in a glass-lined 96-well plate and loadedwith appropriate amounts of test and control solutions for a duration ofa 3-minute exposure at each concentration. Test compound was diluted in0.3% DMSO. An automated parallel patch clamp system, QPatch HT (SophionBioscience A/S, Denmark), was used to evaluate at various concentrations(e.g., 10 μM). The IC₅₀ values was estimated based on the hERGinhibition data. The study was performed at ChanTest (14656 Neo Parkway,Cleveland, Ohio). The QPatch screen is further described by Janzen andBernasconi (eds.), High Throughput Screening, Methods and Protocols,Second Edition, vol. 565, chapter 10, pg. 209-223, 2009.

Selected compounds of formula I were tested in the hERG assay. CompoundsI-1, I-2, I-3, and I-4 gave hERG IC₅₀ of 15.6 uM, 30 uM, 14.6 uM, and13.7 uM, respectively. Compound I-5 shows no observable activity in thehERG assay at 10 uM (no IC₅₀ available). Compound I-6 shows littleobservable hERG activity (<20% inhibition) at 10 uM (no IC₅₀ available).Compound I-7 shows hERG activity (65% inhibition) at 10 uM (no IC₅₀available). Comparator compound I^(C), shown above, produced a hERG IC₅₀of 1.18 uM or activity (87% inhibition) at 10 uM.

Example 14 GSH Trapping in Human Liver Microsome:Protocol

Test compound (final concentration 10 uM) is incubated with either humanor rat liver microsomes (final concentration 1 mg/mL), along withactivating cofactors NADPH (final concentration 1 mM), potassiumphosphate (final concentration 100 mM pH 7.4), magnesium chloride (finalconcentration 3.3 mM) and the trapping agent GSH (final concentration 5mM). The incubation mixture is incubated for 60 min at 37° C. andterminated with ice cold acetonitrile (equal volume as incubationmixture) and the supernatents isolated. The supernatants are eitherinjected directly for LC/MS/MS analysis or dried under N₂ andreconstituted in water:acetonitrile (80:20) mixture before LC/MS/MSanalysis. The corresponding GSH conjugate is evaluated via LC/MS/MS,using a Triple TOF5600/Xevo Qtof MSe.

Example 15 Rat Collagen-Induced Arthritis Model

The collagen induced arthritis (CIA) model in female Lewis rats requiresprimary T and B cell immune responses to type II collagen (CII)immunization for the development of a severe inflammatory disease (seeGoldschmidt T J, Holmdahl R. Cell Immunol. 154(1):240-8, 1994; Helfgott,S. M., et al; Clin. Immunol. Immunopathol. 31:403, 1984; Holmdahl R. etal., J Autoimmun. 7(6):739-52, 1994; and Stuart, J. M., et al., J. Exp.Med. 155:1, 1982). Clinical disease onsets after a secondary CIIchallenge and the disease progresses over the following eight days.

Generally, female Lewis rats are immunized with bovine collagen type IIin incomplete Freund's adjuvant. Rats (N=10/group) receive daily oraladministration of test compound or vehicle BID by oral gavage beginningon day 1 (therapeutic). Clinical severity of arthritis is assessed bycaliper measurements of ankles taken every day beginning on Day 0.

Detailed protocol: Female Lewis rats are immunized subcutaneously withbovine collagen type II (1:1 emulsion of 2 mg/ml bovine CII in 0.01 Nacetic acid: Incomplete Freund's Adjuvant) at three sites of back skin.Six days post immunization rats receive a second subcutaneous injectionof bovine CII. A compound of formula I suspension or vehicle (0.5% CMC,0.1% Tween 80) is administered by oral gavage BID beginning on day 0(prophylactic) (n=10 animals/group). Clinical severity of CIA isassessed by caliper measurements of ankles taken every day beginning onDay 9. Baseline ankle caliper measurements are taken and confirmed asclinically normal (0.260-0.264 in) for prophylactic treatment. Baselineankle caliper measurements for established disease animals is assessedon day 1 of therapeutic dosing and animals are randomly assigned totreatment groups after confirmation of clinical disease onset(0.2751-0.2755 in). Data are analyzed across all groups using a one-wayanalysis of variance (1-way ANOVA), along with an appropriate multiplecomparison post-test. Significance for all tests is set at p<0.05.

Example 16 Analysis of BCR Pathway Activation Via Inhibition ofPhosphorylation of PLCγ2

Protocol: One day before treatment, Ramos cells are plated at a densityof 3×10⁵ cells per well in 200 μL of complete medium in a 96-well tissueculture filter plates (Millipore, Billerica, Mass.). On the day oftreatment, used medium is removed by filtration and the cellsre-suspended in 200 μL serum free medium containing serial compounddilutions and DMSO to 0.1%, then incubated for 2 hours at 37° C. Cellsare stimulated for 5 minutes with 10 μg/mL goat anti-human IgM at 37° C.All medium is removed by filtration and the cells are rinsed with icecold PBS then lysed on ice for 1 hour with lysis buffer containing; 20mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na₃VO₄, 1%Triton X-100, 0.1% SDS, protease inhibitor cocktail, 1 mMphenylmethylsulfonyl fluoride, (PMSF), Phosphatase inhibitor mix 2(Sigma cat #P5726 from Sigma, St. Louis, Mo.), and Phosphatase inhibitormix 3 (Sigma cat #P0044 from Sigma, St. Louis, Mo.). Lysates aresubsequently transferred to standard MSD plates (Meso Scale Discovery,(MSD), (Gaithersburg, Md.)), pretreated with capture antibody(anti-total PLCγ2 antibody B10, (SantaCruz Biotechnologies (Santa Cruz,Calif.)) and blocked with BSA according to the manufacturer'sdirections. Lysates are incubated in the prepared MSD plates overnightat 4° C. with gentle agitation. Wells are washed three times with TBSTand treated with anti pPLCγ2 (SantaCruz) in 1% BSA in PBS for 1 hour atroom temperature. Wells are again washed three times with TBST andtreated with anti-rabbit sulfo-tag antibody (MSD), for 1 hour at roomtemperature. After washing with TBST, MSD read buffer is added and theluminescence is measured in an MSD SECTOR Imager 6000. Maximum responseis determined as the average luminescence in wells containing stimulatedcells treated with anti-IgM and DMSO. Minimal response is determined asthe average luminescence in wells containing unstimulated cells treatedwith DMSO alone. The maximal and minimal values are used to normalizeluminescence in compound treatment wells. The normalized values areplotted against compound concentration on a log scale then analyzedusing Prizm software (GraphPad Software, Inc.). A sigmoidaldose-response equation with variable slope is used to fit the data andgenerate 50% inhibition concentration (IC₅₀).

Ramos cells are incubated in 96 well plates with a range ofconcentrations of a compound of formula I for 2 hours, stimulated with10 μg/mL anti-IgM for 5 minutes, and PLCγ2 phosphorylation measuredusing an electrochemical-luminescent immunoassay. The EC₅₀ is calculatedusing GraphPad Prism software.

Example 17 Inhibition BCR-Induced Human B Cell Proliferation

Human CD19+ B cells are stimulated with an anti-IgM antibody and theactivity of a compound of formula I is evaluated in terms of alteringcellular metabolism after 72 hours. In this context, cellular metabolismdirectly correlates with cellular activation and proliferation, and canalso reflect relative cell survival during proliferation. Anti-IgMantibody is evaluated for effects on B cell proliferation and determinedto exhibit a half-maximal concentration for activation of 10 μg/ml.Using these activation conditions, varying concentrations of testcompound are assayed, in triplicate in 0.1% DMSO, for impact on cellularmetabolism of CD19+ B cells isolated from different donors.

Protocol: Human B cells are isolated from peripheral blood mononuclearcells or unpurified buffy coats using Ficoll-Hypaque gradients(Amersham) and negatively selected by magnetic cell sorting (Human BCell Isolation Kit II, Miltenyi Biotec). Target cell purity isdetermined by flow cytometry by staining for markers of B cells, T cellsand monocytes (CD19, CD3, CD14, respectively; BD Biosciences). Data arecollected on a FACsCaliber flow cytometer and analyzed using FloJosoftware (BD Biosciences). Purity of human B cell preparations isroutinely greater than 95%. Negatively selected human B cells arestimulated with 10 g/mL anti-IgM F(ab′)₂ (Jackson ImmunoResearch) in 96well plates. 100,000 B cells in 0.2 mL RPMI+10% FBS are treated withvarying concentrations (titrated from 5000 nM to 0 nM in 0.5% DMSO) of acompound of formula I in triplicate wells or vehicle control in 0.5%DMSO final concentration for 30 minutes at 37° C., 5% CO₂, then cellsare stimulated with 10 pg/mL anti-IgM F(ab′)₂. B cells are stimulatedfor 72 hr at 37° C., 5% CO₂. Proliferation is measured using theCellTiter-Glo reagent (Promega), as measured on a luminometer. Meanvalues are plotted against maximum proliferation and IC₅₀ values aredetermined using GraphPad Prism v5 software.

Example 18 Evaluation of the Effect of Compounds on Myeloid CellActivation In Vitro

FcγR activation of primary human macrophages. Autoantibody andimmune-complex mediated activation through FcγR can be modeled byactivation of macrophages with immobilized IgG. Primary humanmacrophages derived from GM-CSF treated monocytes up-regulate activationmarkers such as CD80, CD86, MHC antigens and the FcγRIII receptor. Humanmonocyte derived macrophages can be activated by plate-bound purifiedhuman IgG. This stimulation crosslinks the FcγRIII receptor and inducesthe secretion of pro-inflammatory cytokines such as TNFα, IL-6, IL1β andMCP-1. Compound of formula I are evaluated for inhibition of cytokineexpression following FcR activation of human macrophages.

Generally, macrophages are cultured in plates previously incubated withpurified IgG then washed. Titrations of test compound (10,000 nM to 0nM) are added to these cultures. Cell culture supernatants are analyzedby ELISA for the expression of TNFα and IL-6.

Protocol: Human monocytes are isolated from buffy coats of healthydonors and negatively selected by magnetic cell sorting (MonocyteIsolation Kit II, Miltenyi Biotec). Purified monocytes are cultured instandard media supplemented with low-IgG FBS and 100 ng/mL GM-CSF for5-7 days to induce macrophage differentiation. Cultured macrophages arestimulated with 100 μg/mL plate-bound purified IgG a titration of testcompound (10 M to 0 nM). Supernatants are collected after 4 hrs and 18hrs and analyzed for TNFα and IL-6, respectively.

Example 19 Efficacy in Mouse Collagen Antibody-Induced Arthritis

This Example relates not only to arthritis, but also evaluates theactivity of autoantibodies and immune complexes in vivo and therefore isrelevant to other inflammatory disorders such as SLE. In thisexperiment, the activity of autoantibodies and immune complexes producea pathological endpoint that is dependent on FcR signalling, and the Fcportion of such antibodies is inhibited by administration of a compoundof formula I.

The collagen antibody-induced arthritis (CAIA) model in female DBA/1mice does not require cognate T and B cell responses for the inductionof inflammation but rather relies on immune effector mechanisms for thedevelopment of clinical disease. A cocktail of four anti-collagen II(CII) specific monoclonal antibodies and immune stimulatorylipopolysaccharide (LPS) administered 3 days after CII specific antibodytransfer promote antibody-Fc-Receptor engagement (Kagari T. et al.; JImmunol. 170:4318-24 (2003)), immune complex formation, complementactivation (Banda N K, et al.; Clin Exp Immunol. 159:100-8 (2010)) andpro-inflammatory cytokine production to induce a severe inflammatorydisease over a 10 day period.

Generally, arthritis is induced by injection of a cocktail of monoclonalanti-collagen antibodies into DBA/1 mice on day 0. Mice (N=10/group)receive daily oral administration of test compound either QD or BID asindicated beginning on day 0. Paw inflammation is evaluated daily.

Protocol: Female DBA/1 mice 6-8 weeks of age receive 2 mg of anarthitogenic four clone monoclonal antibody cocktail (Chondrex #10100)i.v. on day 0 followed by a 50 ug dose of LPS on 3 days later. Testcompound suspension or vehicle (0.5% CMC, 0.1% Tween 80) is administeredBID by oral gavage beginning on day 0 (10 animals/group) just prior toi.v. transfer of antibody cocktail. Clinical severity of CIA is assessedby monitoring inflammation on all four paws, applying a scale rangingfrom 0 to 4. Each paw is graded as follows: 0, normal; 1, mild butdefinite redness and swelling of the ankle or wrist, or redness andswelling of any severity for 1 or 2 digits; 2, moderate to severeredness and swelling of the ankle or wrist, or more than two digits; 3,redness and swelling (pronounced edema) of the entire paw; and 4,maximally inflamed limb with involvement of multiple joints. The sum ofthe four individual scores is the arthritis index, with a maximalpossible score of 16 for each animal.

1. A pharmaceutical salt of formula I:

wherein: each R¹ is independently hydrogen, an optionally substitutedC₁₋₆ aliphatic group, an optionally substituted 3-7 membered monocyclicheterocyclic group, or an optionally substituted heterocyclylalkyl grouphaving 3-7 carbon atoms and 1-3 heteroatoms independently selected fromnitrogen, oxygen, and sulfur; or two R¹ groups are taken together withtheir intervening atoms to form an optionally substituted 3-7 memberedsaturated or partially unsaturated monocyclic heterocyclic ring having1-2 heteroatoms independently selected from nitrogen, oxygen, andsulfur; wherein optionally substituted groups may be substituted withhalogen, —NO₂, —CN, —OR, —SR, —N(R)₂, —C(O)R, —CO₂R, —N(R)C(O)OR,—C(O)N(R)₂, —OC(O)R, —N(R)C(O)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; each Ris independently hydrogen or C₁₋₆ aliphatic; or two R groups attached tothe same nitrogen are taken together with their intervening atoms toform an optionally substituted 3-7 membered saturated or partiallyunsaturated monocyclic heterocyclic ring having 1-2 heteroatoms, inwhich any second heteroatom is independently selected from nitrogen,oxygen, and sulfur; Ring A is

R² is —Cl or —F; and R³ is —CF₃, —OCF₃, or —F.
 2. The pharmaceuticalsalt of claim 1, wherein the compound is of formula II-a:


3. The pharmaceutical salt of claim 1, wherein the compound is offormula II-b:


4. The pharmaceutical salt of claim 1, wherein the compound is offormula III:


5. The pharmaceutical salt of claim 1, wherein the compound is formulaIV:


6. The pharmaceutical salt of claim 5, wherein R³ is —CF₃ or —F. 7.(canceled)
 8. The pharmaceutical salt of claim 5, wherein both R¹ arehydrogen.
 9. The pharmaceutical salt of claim 5, wherein one R¹ ishydrogen and the other R is an optionally substituted C₁₋₆ aliphatic.10. The pharmaceutical salt of claim 9, wherein one R¹ is hydrogen andthe other R¹ is methyl.
 11. The pharmaceutical salt of claim 5, whereinboth R¹ are optionally substituted C₁₋₆ aliphatic groups.
 12. Thepharmaceutical salt of claim 1, wherein one R¹ is hydrogen and the otheran is optionally substituted C₁₋₆ aliphatic.
 13. The pharmaceutical saltof claim 1, wherein both R¹ are optionally substituted C₁₋₆ aliphaticgroups.
 14. The pharmaceutical salt of claim 1, wherein both R¹ arehydrogen. 15.-19. (canceled)
 20. A pharmaceutical salt selected from thegroup consisting of:


21. A method of decreasing the enzymatic activity of Bruton's tyrosinekinase comprising contacting Bruton's tyrosine kinase with an effectiveamount of a pharmaceutical salt of claim 1 or a composition thereof. 22.A method of treating a disorder responsive to inhibition of Bruton'styrosine kinase comprising administering to a subject an effectiveamount of a pharmaceutical salt of claim 1 or a composition thereof. 23.A method of treating a disorder selected from the group consisting ofautoimmune disorders, inflammatory disorders, and cancers comprisingadministering to a subject an effective amount of a pharmaceutical saltof claim 1 or a composition thereof.
 24. The method of claim 23, whereinthe disorder is rheumatoid arthritis, systemic lupus erythematosus, oratopic dermatitis. 25.-26. (canceled)
 27. The method of claim 23,wherein the disorder is leukemia or lymphoma.
 28. A pharmaceuticalcomposition comprising a pharmaceutical salt of claim 1 and one or morepharmaceutically acceptable excipients.